Anal. Chem. 2001, 73, 1146-1154
Amperometric Ion Detection in Capillary Zone Electrophoresis by Ion Transfer across a Liquid-Liquid Microinterface Stefan Wilke,* Ronald Schu 1 rz, and Hanming Wang†
Department of Chemistry (Merseburg), Martin-Luther-University Halle-Wittenberg, D-06099 Halle, Germany
Ion transfer across the electrically charged interface of two immiscible electrolytes has been employed for the amperometric detection of ions in capillary zone electrophoresis (CE). The method is inherently selective to hydrophobic ions, which are characterized by a low standard Gibbs energy of partition between the aqueous CE buffer solution and 2-nitrophenyl n-octyl ether employed as organic phase. The applicability of this detection scheme for nonredox ions is demonstrated by means of the cations choline and acetylcholine and by some alkyl and aryl sulfate and sulfonate anions. The response of the detector is sufficiently fast; no additional peak broadening has been observed. Plate numbers of 100 000 (170 000 m-1) were determined for the quickly migrating choline cation. It is shown that the use of buffer modifiers such as organic solvent additives, cyclodextrins, and polyelectrolytes is compatible with the detector within certain boundaries. The limit of detection was ∼1 µM for the ions tested.
Capillary electrophoresis (CE) has proved to be a powerful separation method for solving a number of problems in analytical and bioanalytical chemistry.1,2 Among the various detection strategies, the techniques based on light absorbance and fluorescence are most popular and are commonly implemented in commercial CE instruments. However, the sensitivity of UVvisible absorbance detection is affected by the short optical path length, so that the sensitivity is significantly worse than in HPLC using the same detection principle. Fluorescence detection can be very sensitive, but is restricted to the relatively few ionic species that are fluorescent or can be labeled with fluorescent markers. In particular, small and mobile ions can be detected conductometrically,3,4 yet the performance can be limited by a large background conductivity of the separation buffer. Amperometric * Corresponding author: (e-mail)
[email protected]; (fax) ++49 (3461) 46-30 43. † On leave from the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China. (1) Li, S. F. Y. Capillary Electrophoresis; Elsevier: New York, 1993. (2) Claire, R. L. S. Anal. Chem. 1996, 68, 568R. (3) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 1. (4) Huang, X.; Pang, T.-K. J.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1987, 59, 2747.
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detection at metal or carbon electrodes is selective to redox-active species and often very sensitive5,6 but inherently not suitable for nonredox species. The latter have been detected by utilizing selfassembled monolayers on gold electrodes7 and also by potentiometry with ion-selective microelectrodes8 based on the partition or transfer of the analyte ions between the aqueous CE separation buffer and the organic or “membrane” phase of the ion-selective electrode. Unfortunately, the voltage signal of ion-selective electrodes often does not depend on the concentration either logarithmically or linearly, and a relatively laborious and more complicated calibration procedure can be required therefore. The electrically induced transfer of ions across the polarized interface between two immiscible electrolytes (ITIES) has been investigated by modern electrochemical techniques for almost thirty years.9-11 Nitrobenzene and 1,2-dichloroethane have been the most popular solvents for studying L-L interfaces. However, for analytical purposes, these relatively volatile, water-soluble and toxic solvents are increasingly replaced by 2-nitrophenyl n-octyl ether (NPOE) and related derivatives.12-14 Unlike conventional (redox) electrochemistry, the faradaic current is the result of the transfer of ions from an aqueous into an organic phase or vice versa. Because of the ionic charge, the mass transfer is accompanied by a charge transfer across the phase boundary, which is measured as cell current in the potentiostatic circuit. By convention, the cell current is positive when positive charges are transported from the aqueous to the organic phase or negative charges in the opposite direction. Doing without electrochemical terminology, ion-transfer voltammetry can also be considered as electrically driven partition or extraction of ions between two immiscible electrolyte solutions, where the partition or distribution coefficient of the ionic species is a function of the electrical potential applied between the two phases. This is expressed quantitatively by Nernst’s equation (5) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762. (6) Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 61, 292A. (7) He, P.; Ye, J.; Fang, Y.; Suzuki, I.; Osa, T. Anal. Chim. Acta 1997, 337, 217. (8) Haber, C.; Silvestri, I.; Ro ¨o ¨sli, S.; Simon, W. Chimia 1991, 45, 117. (9) Koryta, J. Electrochim. Acta 1988, 33, 189. (10) Vany´sek, P. Anal. Chem. 1990, 62, 827A. (11) Girault, H. H. Mod. Aspects Electrochem. 1993, 25, 1. (12) Valent, O.; Koryta, J.; Panoch, M. J. Electroanal. Chem. 1987, 226, 21. (13) Sawada, S.; Osakai, T.; Senda, M. Anal. Sci. 1995, 11, 733. (14) Osborne, M. D.; Girault, H. H. Electroanalysis 1995, 7, 714. 10.1021/ac0011890 CCC: $20.00
© 2001 American Chemical Society Published on Web 02/10/2001
∆wo φ ) ∆wo φo +
RT coγo ln zF cwγw
(1)
where co, cw, γo, and γw are the respective concentrations and activity coefficients in the organic (o) and aqueous (w) phase; ∆wo φ is the actual Galvani potential difference across the L-L interface, z is the charge number of the transferred ion, and R, T, and F have their usual meaning. The standard potential ∆wo φo is the electrochemical expression for the standard Gibbs energy of ion partition ∆ow Gop between water and the organic phase according to
∆ow Gop ) zF∆wo φo
(2)
In most experimental cases, the kinetics of the ion transfer can be considered as electrochemically reversible; i.e., the fluxes and therefore the current are essentially limited by the transport of ions toward and away from the interface. The diffusion scheme at the L-L interface is comparable with a mercury electrode, where the oxidized and reduced forms are diffusing as dissolved particles in the electrolyte and in the Hg phase, respectively. In general, the well-known voltammetric formalisms can be transferred directly to ion-transfer voltammetry. An introductory review on principles and applications of ion transfer across L-L interfaces has been given, for example, by Vany´sek.10 Fundamental investigations on L-L interfaces were always, more or less emphatically, accompanied by ideas and attempts to use the ion transfer as an electrode process for the voltammetric analysis of ions that cannot be accessed by common redox electrochemistry.14-27 The determination of dodecyl sulfate in a tooth salt,15 volatile amines in fish,16 K+ and Na+ in mineral water17 and human blood serum,18 and nitrate in river water 19 are examples of successful attempts to employ polarized L-L interfaces for the determination of ions and ionizable molecules in realworld samples. L-L interfaces were also used to detect ions in flow injection analysis 18-23 and HPLC.23-25 The transfer of sulfonated hydrocarbons of various chain lengths across the electrically charged water-nitrobenzene and water-dichloroethane interface has been investigated mainly by Suzuki et al.28 In recent years, miniaturized L-L microinterfaces (LLM) have been developed and employed for basic investigations as well as for analytical purposes. In this work, LLM were prepared as (15) Krause, J.; Umland, F. Fresenius Z. Anal. Chem. 1989, 335, 791. (16) Yamamoto, Y.; Nuno, T.; Osakai, T.; Senda, M. Bunseki Kagaku 1989, 38, 589. (17) Yamamoto, Y.; Osakai, T.; Senda, M. Bunseki Kagaku 1990, 39, 655. (18) Hundhammer, B.; Solomon, T.; Zerihun, T.; Abegaz, M.; Bekele, A.; Graichen, K. J. Electroanal. Chem. 1994, 371, 1. (19) Wilke, S.; Franzke, H.; Mu ¨ ller, H. Anal. Chim. Acta 1992, 268, 285. (20) Marecˇek, V.; Ja¨nchenova, H.; Colombini, M. P.; Papoff, P. J. Electroanal. Chem. 1987, 217, 213. (21) Hundhammer, B.; Wilke, S. J. Electroanal. Chem. 1989, 266, 133. (22) Sawada, S.; Torii, H.; Osakai, T.; Kimoto, T. Anal. Chem. 1998, 70, 4286. (23) Wang, E.; Ji, H. Electroanalysis 1989, 1, 75. (24) Wilke, S.; Wang, H. M.; Muraczewska, M.; Mu ¨ ller, H. Fresenius J. Anal. Chem. 1996, 356, 233. (25) Lee, H. J.; Girault, H. H. Anal. Chem. 1998, 70, 4280. (26) Osborne, M. D.; Girault, H. H. Mikrochim. Acta 1995, 117, 175. (27) Lee, H. J.; Beriet, C.; Girault, H. H. J. Electroanal. Chem. 1998, 453, 211. (28) Suzuki, M.; Kihara, S.; Maeda, K.; Ogura, K.; Matsui, M. J. Electroanal. Chem. 1990, 292, 231.
reported by Campbell and Girault.29 Two immiscible electrolyte solutions were separated by using a few micrometers thick film of polyester, in which a microhole of ∼20-µm diameter was drilled using a laser ablation technique. A flat LLM is formed at the microhole due to the interfacial tension. The response of the microhole-supported LLM toward ions in the aqueous phase has been shown to be similar to that of an inlaid microdisk electrode.30 An important advantage is that usually the IR drop can be ignored. Both individuals and ensembles of LLM have been utilized for the preparation of enzymatic urea14 and creatinine26 sensors, for the stripping voltammetric determination of choline,31 and for the flow-through amperometric detection of ions.24,27 The basic idea for starting the present work was that a single LLM of ∼10-50-µm diameter could be suitable for serving as an amperometric microprobe for end-column detection in CE. The detector would inherently be selective to ions of a sufficiently high lipophilicity and could possibly complement the application of other CE detectors. As for amperometry with conventional electrodes, a linear dependence between detector current and analyte concentration, a lower detection limit than for potentiometry, and a fast response was anticipated. Particular attention has been devoted to anionic surfactants in this work, which is why a brief review should be given here. The separation of anionic surfactants by means of CE has been subject of a number of papers (for a review, see ref 32). For example, alkyl sulfonate and sulfate surfactants have been separated by Chen and Pietrzyk,33 Shamsi and Danielson,34-36 and Heinig et al.37 Vogt et al. investigated the separation and determination of linear alkylbenzenesulfonates (LAS) and demonstrated that the separation of homologues and isomers of LAS can be achieved by modifying the CE separation buffer with acetonitrile and cyclodextrins.38,39 In particular, for long-chain surfactants, modification of the buffer and the sample with organic solvents such as methanol and acetonitrile has been found necessary to minimize adsorption of the solutes onto the wall of the capillary. Most measurements, in particular of aromatic sulfonates, have been made using direct UV photometric detection (e.g., refs 33 and 38-40). Nonaromatic anionic surfactants such as alkyl sulfates and sulfonates have been detected by indirect UV photometry,33,35 direct41 and indirect42 conductometry, potentiometry with ionselective electrodes,43,44 and electrospray mass spectrometry.45 (29) Campbell, J. A.; Girault, H. H. J. Electroanal. Chem. 1989, 266, 465. (30) Osborne, M. C.; Shao, Y.; Pereira, C. M.; Girault, H. H. J. Electroanal. Chem. 1994, 364, 155. (31) Lee, H. J.; Beriet, C.; Girault, H. H. Anal. Sci. 1998, 14, 71. (32) Vogt, C.; Heinig, K. Fresenius J. Anal. Chem. 1999, 363, 612. (33) Chen, S.; Pietrzyk, D. J. Anal. Chem. 1993, 65, 2770. (34) Shamsi, S. A.; Danielson, N. D. Anal. Chem. 1994, 66, 3757. (35) Shamsi, S. A.; Danielson, N. D. Anal. Chem. 1995, 67, 4210. (36) Shamsi, S. A.; Danielson, N. D.; Warner, I. M. J. Chromatogr., A 1999, 835, 159. (37) Heinig, K.; Vogt, C.; Werner, G. J. Capillary Electrophor. 1996, 3, 261. (38) Vogt, C.; Heinig, K.; Langer, B.; Mattusch, J.; Werner, G. Fresenius J. Anal. Chem. 1995, 352, 508. (39) Heinig, K.; Vogt, C.; Werner, G. J. Chromatogr., A 1996, 745, 281. (40) Zweigenbaum, J. Chromatogram 1990, 11, 9. (41) Gallagher, P. A.; Oertel, C. M.; Danielson, N. D. J. Chromatogr., A 1998, 817, 31. (42) Gallagher, P. A.; Danielson, N. D. J. Chromatogr., A 1997, 781, 533. (43) Schnierle, P.; Kappes, T.; Hauser, P. C. Anal. Chem. 1998, 70, 3585. (44) Poels, I.; Nagels, L. J. Anal. Chim. Acta 1999, 385, 417. (45) Tanaka, Y.; Kishimoto, Y.; Otsuka, K.; Terabe, S. Bunseki Kagaku 1998, 47, 563.
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microhole at the center, was sealed onto the lower orifice of a glass tube (7-mm i.d., 10-mm o.d., 40-mm length) by silicon rubber (product 494-118, RS Components). After the silicon rubber was allowed to cure for 24 h, the glass tube was filled with ∼250 µL of NPOE containing 10 mM TDATPBCl as supporting electrolyte. The glass tube with the polymer film was immersed into the detection cell filled with the CE buffer solution. Glass tube and cell body were positioned in such a way that the microhole was faced with the outlet orifice of the CE capillary, with a distance of 50 µm between them. A silver wire, mantled by Teflon tubing, was oxidized in a saturated solution of K2S2O8 for ∼1 h and then conditioned in the organic electrolyte solution for 1 day, to serve as “organic”, junction-free reference electrode in the organic phase. We assume that a layer of AgTPBCl is formed on the Ag wire, and a Ag/AgTPBCl(s), TDATPBCl reference electrode is obtained. A double-junction Ag/AgCl(s), KCl (saturated) reference electrode with two Vycor diaphragms was employed in the aqueous phase. Platinum wires served as auxiliary electrode in the organic phase and as electrode in the inlet vial (anode of the CE system). The electrochemical cell can be written as Figure 1. Scheme of the CE setup with the L-L microinterface as amperometric detector: (1) inlet buffer reservoir; (2) separation capillary; (3) polymer film with the microhole; (4) glass tube; (5) aqueous electrolyte solution in the detector cell; (6) organic electrolyte solution; (7) auxiliary electrode and (8) reference electrode in the organic electrolyte solution; (9) Ag/AgCl reference electrode in the aqueous phase; (10) detector cell body of stainless steel.
Very low detection limits have been achieved by direct46,47 and indirect36 fluorescence detection. EXPERIMENTAL SECTION Chemicals. Aqueous solutions were prepared using ultrapure water (18 MΩ cm) from an Elgastat UHQ purification system (Elga Ltd., High Wycombe, Bucks, England). NPOE and tetradodecylammonium tetrakis(p-chlorophenyl)borate (TDATPBCl) were of Selectophore grade (Fluka, Neu-Ulm, Germany). NPOE was purified by passing it slowly through a 3-cm-long column of neutral activated alumina.48 The separation buffer (pH 10, ∼13 mM sodium tetraborate/∼18 mM NaOH, Fluka product) was pressed through a 0.45-µm syringe filter and ultrasonicated before use. Industrial surfactant mixtures were provided by various manufacturers: Marlon PS 65 and Marlon A 390 (Hu¨ls, Marl, Germany), Texapon SB 3 KC (Henkel, Du¨sseldorf, Germany), Jordapon Cl Prilled (PPG Industries), and TEGO Betain L5040 (Th. Goldschmidt, Germany). L-L Microinterface Amperometric Detector. A scheme of the end-column49 detector connected to the CE system is given in Figure 1. Microholes were drilled into a 35-µm-thick film of poly(ethylene terephthalate) (Melinex “S”, ICI Films) using a laser ablation technique.30,50 The diameter of the microholes was 31 µm unless otherwise stated. A disk of this film, with a single (46) Kanz, C.; Nolke, M.; Fleischmann, T.; Kohler, H. P. E.; Giger, W. Anal. Chem. 1998, 70, 913. (47) Loos, R.; Niessner, R. J. Chromatogr., A 1998, 822, 291. (48) Samec, Z.; Langmaier, J.; Trojanek, A. J. Electroanal. Chem. 1996, 409, 1. (49) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189. (50) Seddon, B. J.; Shao, Y.; Girault, H. H. Electrochim. Acta 1994, 39, 2377.
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Ag/AgCl(s), KCl (sat)//aq test electrolyte/*10 mM TDATPBCl in NPOE, AgTPBCl(s)/Ag′
where the interface of interest, i.e., the working electrode, is indicated by an asterisk. The experimentally accessible voltage E between the reference electrodes
E ) φAg - φ′Ag ) ∆wo φ + Ewref - Eoref
(3)
comprises the Galvani potential difference ∆wo φ across the L-L interface and the potentials of the aqueous and organic reference electrodes, Ewref and Eoref, respectively. The steel body of the detection cell was electrically grounded and served simultaneously as aqueous auxiliary electrode of the detector and as cathode of the CE circuit. The reference and auxiliary electrodes in the aqueous and organic phases were connected to a four-electrode potentiostat, which is shown schematically in Figure 2. No compensation for the IR drop was applied. The instrumentation amplifiers (INA111AP and INA116AP) and the control amplifier (OPA177GP) were obtained from Burr Brown (Tucson, AZ). An A/D interface was employed for sampling the data and sending them to a PC. To show electrophoreograms with baselines close to the abscissa (zero current) in the figures, the mean base current Ib was subtracted from the electrophoreogram in each case. Electric Field Decoupler. A decoupler was manufactured by mounting together the 594-mm-long separation capillary and a 6-mm-long piece of the “decoupling” capillary by mantling the connection with a 4-mm-long jacket of a porous hollow fiber of polyacrylonitrile. After mounting them together, the porous jacket was soaked with 2-hydroxyethyl methacrylate previously saturated with dibenzoyl peroxide. The outer surface of the hollow fiber was then wetted with N,N′-dimethyltoluidine, which started the polymerization of the acrylate. Air was pressed through the capillary to prevent plugging of the capillary by the possibly
Figure 2. Scheme of the electronic circuit of the four-electrode potentiostat. Ro and Rw are the reference electrodes in the organic and in the aqueous phase, respectively, and Ao and Aw are the auxiliary electrodes in the respective phases.
penetrating acrylate monomer. Before use, the decoupler was stored in the electrophoretic buffer to remove soluble, potentially interfering components such as N,N′-dimethyltoluidine from the decoupler and to allow the penetration of the buffer solution into the polymer. The decoupling effect is based on the fact that the electrical resistance through the short “decoupler capillary” is sufficiently higher than the short path through the capillary gap and the hollow fiber jacket. CE System. Measurements were performed using a commercial CE system (Prince Technologies, Emmen, Netherlands), comprising autosampler, injection unit, and high-voltage (HV) power supply. This system was selected, since pressure or vacuum can be applied to the inlet of the capillary, while the outlet of the capillary is freely available for the end-column electrochemical detector. Samples were injected by the application of pressure (40 or 120 mbar for 6 s, i.e., 6.1- or 18.3-nL volume, 3.1- or 9.4mm plug length) to the inlet vial. The separation voltage was 25 kV. The polyimide-covered capillaries of fused silica (CS Chromatographie-Service Langerwehe, Germany) were 60 cm in length and 50-µm i.d. (365-µm o.d.). Before the first use, the capillaries were rinsed with background electrolyte overnight. Rinsing with NaOH was avoided, since this led to a strong tailing of some peaks. The capillary was flushed with buffer for 30 s and 1 bar pressure immediately before each injection. Standard solutions were prepared in pure distilled water. All measurements were carried out at laboratory temperature (23 ( 3 °C). The capillary was not actively cooled, since its main section was placed outside the thermostat chamber in the “free” air. Alignment of the L-L Microinterface. An x-y-z micropositioner was employed to change the position of the end of the capillary, whereas the cell with the LLM remained fixed. The following procedure was performed to find out the position where
the LLM exactly matches the axis of the capillary. First, a small but still visible gap (100-200 µm) was adjusted between the end of the capillary and the polymer film with the LLM. Then, the radial position between the capillary and the LLM was aligned by one of two different methods, dependent on whether a decoupler was used or not. Utilizing an electric field decoupler, a solution of 0.1 mM sodium dodecylbenzenesulfonate in borate buffer was pumped through the capillary by applying 1 bar pressure. The working potential was fixed in or before the limiting diffusion current region, and the detector current I was monitored. The position of the capillary outlet was varied in parallel to the plane of the polymer film of the cell until |I| reached a maximum. In measurements without decoupler, the best position was conveniently found by employing the (ideally hemispheric) potential gradient of the HV at the capillary outlet. For this purpose, the potentiostatic control loop was broken, and the voltage between the aqueous and organic reference electrodes was monitored as in potentiometry. Similar to the previous procedure, the position of the capillary outlet was varied until a maximum crossover of the HV on the potential between the reference electrodes was observed. Common to both cases, a distance of 50 µm between the orifice of the capillary and the LLM was adjusted then. The “zero distance” point, when the end of the capillary touches the polymer film, was obtained by slowly driving the capillary against the polymer film. Simultaneously, the surface of the polymer film was observed against the border between a black and a light background. When the capillary touched the film, it was slightly pushed in, and the image reflected by the polymer film changed. The reproducibility of this method was ∼5 µm, which corresponds with the scaling of the micropositioner and is satisfactory for a total distance of 50 µm. Finally, the crossover of the HV field at the LLM was measured by monitoring the potential change between the two reference electrodes when the HV is switched between “on” and “off”. The potentiostat was switched off for this procedure. A pressure of 1 bar was applied to the inlet buffer vial, to avoid the effect of the electroosmotic flow (EOF) on the measured potential when the HV is applied. Without decoupler, the crossover of the high-voltage field was usually on the order of (+400 ( 50) mV. The particular value was taken into consideration when the working potential was chosen. Employing the decoupler, the HV crossover was below 20 mV, and no such correction was applied. RESULTS AND DISCUSSION General Characterization of the Detector Response to ACh+/Ch+. Since Ch+ and ACh+ are relatively mobile model ions, which comigrate with the EOF, they were chosen to test the dynamic response of the detector. A cyclic voltammogram for the transfer of choline and acetylcholine across a water-NPOE microinterface of 31-µm diameter is, after subtraction of the voltammogram of the base electrolyte, shown in Figure 3. When the potential is changing in the positive direction, a wave for the ion transfer from the aqueous into the organic phase is observed for both ions. This behavior is typical for microelectrodes, when the scan rate and the electrode diameter are sufficiently small. A peak-shaped signal for the backward scan reveals a planar component of diffusion in the organic phase. From the point of view of the ions in the organic phase, the LLM is a recessed microdisk electrode at which ion diffusion is both planar (inside Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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To keep Figure 4 as clear as possible, the time scale of each electrophoreogram was manipulated in such a way that, after the recalculation, the maximum of each Ch+ peak exactly matched the average value. This was accomplished by applying the transformation
1 1 1 1 ) + t t ˆt i ˆt max i max
(4)
where ˆti, ti, ˆtmax, and tmax are the recalculated time data, the real time data, and the preset (here 127.0 s) and the really measured time of the peak maximum, respectively. Using the equation
µi ) Figure 3. Baseline-corrected cyclic voltammograms of 0.1 mM ACh+ (s) and Ch+ (- - -), and voltammogram of the base electrolyte 5 mM Li2SO4 (‚‚‚). Sweep rate, 8 mV/s.
(
l2 1 1 V tp,i tEOF
)
where l, V, and tp,i are the length of the capillary, the separation high voltage, and the migration time of the ion i, respectively, the net mobilities µi of Ch+ and ACh+ were determined to be 0.400 and 0.351 cm2/kV s (RSD
