Anal. Chem. 2006, 78, 4709-4712
Kinetic Monitoring of Electrophoretically Induced Solute Reaction by Axial Absorption Detection with Liquid-Core Waveguide Akira Wada, Makoto Harada, and Tetsuo Okada*
Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan
Teflon AF-2400 capillary has been used for capillary electrophoretic separation as well as for liquid-core waveguide for axial absorption detection. This separation/ detection scheme has allowed continuous monitoring of electrophoretically induced reactions. In this paper, the decomposition of Cd2+ complex with 4-(2-pyridylazo)resorcinol has been tested, and its decomposition kinetics has been studied. A simple modeling has predicted the single-exponential decay of the absorbance detected by the present axial absorption detector and has allowed the estimation of the decomposition rate constant for this reaction. Capillary electrophoresis (CE) is a powerful tool for separation of various classes of compounds, including inorganic ions, organic molecules, polymers, and particles and has found various applications.1-4 Terminal detectors with optical, electrochemical,5,6 and mass spectrometric methods7-9 have been usually employed to monitor the migration and separation of solutes. This useful and versatile detection scheme has been widely applied to stable solutes that do not change their chemical forms during a CE separation process. In contrast, a terminal detector does not give a signal to an unstable solute that is decomposed before it reaches the detector. Labile complexes, for example, which are present under equilibria with free ligands, will be decomposed if the electrophoretic mobility of the complexes is different from those of the ligands and give no signals to a terminal detector. Detection of such complexes is possible only when excess ligands are added to the running buffer. Such a way has been often used to separate metal ions with on- or off-capillary complexation.10 Iki et al. have devised an entirely different strategy, and successfully developed kinetic CE, in which metal complexes with 4-(2-pyridylazo)resorcinol (PAR) were electrophoretically separated with the * To whom correspondence should be addressed. Phone and Fax: +81-35734-2612. E-mail:
[email protected]. (1) Powell, P. R.; Ewing, A. G. Anal. Bioanal. Chem. 2005, 382, 581. (2) Simo, C.; Barbas, C.; Cifuentes, A. Electrophoresis 2005, 26, 1306. (3) Kremser, L.; Blaas, D.; Kenndler, E.Electrophoresis 2004, 25, 2282. (4) Shintani, T.; Torimura, M.; Sato, H.; Tao, H.; Manabe, T. Anal. Sci. 2005, 21, 57. (5) Wang, J. Electroanalysis 2005, 17, 1133. (6) Shin, D.; Sarada, B. V.; Tryk, D. A.; Fujishima, A.; Wang, J. Anal. Chem. 2003, 75, 530. (7) Michalke, B. Electrophoresis 2005, 26, 1584. (8) Monton, M. R. N.; Terabe, S. Anal. Sci. 2005, 21, 5. (9) Schmitt-Kopplin, P.; Englmann, M. Electrophoresis 2005, 26, 1209. (10) Padarauskas, A. Electrophoresis 2003, 24, 2054. 10.1021/ac060175c CCC: $33.50 Published on Web 05/16/2006
© 2006 American Chemical Society
running solution not containing PAR.11-13 Labile complexes, which are formed in the presence of an excess ligand, were decomposed during CE separation and did not give detector responses, while inert ones were still detected with a terminal spectrophotometric detector even in the absence of the ligand. This scheme allowed the selective disappearance of Mn2+-, Cd2+-, and Zn2+-PAR complexes. Although this is a very interesting and potentially useful method, its obvious disadvantage is that the selective detection of labile complexes is not possible. One of the most important applications of CE is to evaluate solution reactions that cannot be probed by other method. Various solution-phase equilibria have been studied by CE, involving complex formation,14,15 hydrogen bond interaction,16 ion association,17 etc. Two major concepts, i.e., electrophoretically mediated microanalysis18,19 and electroinjection analysis,20,21 have been devised to probe the kinetic aspects of solution-phase reactions by CE. In these methods, two zones encounter each other somewhere inside the capillary, and reaction products are usually detected by a terminal detector. Small reaction space and separation functionality of CE have allowed us to probe on-capillary derivatization and kinetics of enzymatic reactions. A limitation of these methods comes from terminal detection, which cannot be used for continuous monitoring of the reaction under study. Whole capillary detection or imaging is a useful technique to visualize electrophoretic separation processes22-23 and is applicable to evaluation of kinetic features of reactions.24 Axial optical detection with a liquid-core waveguide (LCW) offers a simpler alternative, which allows the continuous monitoring of the events occurring in the capillary during electrophoretic processes. Although LCW has been applied to fluorescence and Raman detection in CE,25-29 (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
Iki, N.; Hoshino, T.; Yotsuyanagi, T. Chem. Lett. 1993, 701. Iki, N.; Hoshino, T.; Yotsuyanagi, T. J. Chromatogr., A 1996, 652, 539. Iki, N. Bunseki Kagaku 2002, 51, 495. Okada, T. J. Chromatogr., A 1995, 695, 309. Bartak, P.; Bednar, P.; Kubacek, L.; Stransky, Z. Anal. Chim. Acta 2000, 407, 327. Okada, T. J. Chromatogr., A 1997, 771, 275. Takayanagi, T. Anal. Sci. 2004, 20, 255, and references therein. Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217. Priego-Capote, F.; Luque de Castro, M. D. Electrophoresis 2004, 25, 4074, and references therein. Andreev, V. P.; Kamnev, A. G.; Popov, N. S. Talanta 1996, 43, 909. Andreev, V. P.; Christian, G. D. Anal. Lett. 2001, 34, 1569, and references therein. Kitagawa, F.; Aizawa, S.; Otsuka, K. Anal. Sci. 2005, 21, 61. Xu, F.; Jabasini, M.; Zhu, B.; Ying, L.; Cui, X.; Arai, A.; Baba, Y. J. Chromatogr., A 2004, 1051, 147. Takahashi, T.; Ohtsuka, K.; Iki, N.; Hoshino, H. Analyst 2005, 130, 1337.
Analytical Chemistry, Vol. 78, No. 13, July 1, 2006 4709
Figure 1. Schematic illustration of an experimental setup.
light absorption detection is also possible, which undoubtedly extends the usefulness of axial detection schemes. Although Xi and Yeung30 already showed that an axial detection scheme can be used for absorbance detection in CE, LCW should allow easy probing of not only electrophoretic separation but also the reaction induced by electrophoretic separation. In the present paper, electrophoretically induced decomposition of a labile complex is monitored by the axial light absorption detection with LCW and is kinetically characterized on the basis of a simple model. EXPERIMENTAL SECTION An experimental setup is schematically illustrated in Figure 1. A Teflon AF-2400 tube (0.508 mm o.d. × 0.254 mm i.d.; Biogeneral) was used for an LCW as well as for CE separation. The typical length of the Teflon AF-2400 tube was 30 cm. The tube was interfaced to an incident (1-mm core) and a detection (0.1-mm core) optical fiber with PTFE “T” connectors. Electrodes for electrophoresis were inserted to the T connectors. To suppress changes in pH of the running buffer, direct contact of the Pt electrode with the running buffer was avoided using a glass tube with a porous glass tip. The Pt electrodes inserted into the glass tubes were connected to a power supply model HCZE-30P No. 25 (Matsusada). Light sources were a W-lamp or LED with an appropriate range of the emission wavelengths. A CCD array spectrophotometer (Ocean Optics) was used as a detector. A sample solution was injected from the anodic end by siphoning for 2-3 s by keeping a sample bottle 5 cm higher than the running position. After a sample was injected, the running buffer was introduced for 2-3 s to prevent the electrophoretic leaking of sample components from the injection end. Although two buffer reservoirs was basically set at the same horizontal positions, their relative heights were adjusted so that a small flow toward the cathode always allowed the elution of injected components from the cathodic end; very small pressurized flow was possibly involved, albeit the flow toward the cathode was predominantly caused by electroosmosis. All of the instruments were installed in an incubator set at 25 °C. A usual capillary electrophoretic system was composed of a fused-silica capillary (50 µm i.d. × 50 cm in length), a UV-visible detector model CE-1570 (Jasco), and the power supply. The running buffer (1 mM Borax and NaH2PO4, pH 8.38) and sample solutions were prepared with MilliQ water. Sample (25) Dasgupta, P. K.; Genfa, Z.; Li, J.; Boring, B.; Jambunathan, S.; Al-Horr, R. Anal. Chem. 1999, 71, 1400. (26) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887. (27) Liu, Z.; Pawliszyn, J. Anal. Biochem. 2005, 336, 94. (28) Kostal, V.; Zeisbergerova, M.; Slais, K.; Kahle, V. J. Chromatogr., A 2005, 1081, 36. (29) Wang, S.; Fang, Z. Anal. Bioanal. Chem. 2005, 382, 1747. (30) Xi, X.; Yeung, E. S. Appl. Spectrosc. 1991, 45, 1199.
4710 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
Figure 2. Change in transmitted light intensity during electrophoresis of Ni2+(PAR)2 complex. (A) Intensity of transmitted light. (B) Differentiation of light intensity with time. Running solution, 1 mM NaH2PO4 + Na2B4O7 (pH 8.38). Sample, 1 µM Ni2+ and 0.5 mM PAR. Length of Teflon AF-2400 tube, 37 cm. Applied voltage, 4 kV.
solutions containing 0.5 mM PAR and metal ion (1 µM Ni2+ or 10 µM Cd2+) were prepared in the same buffer. RESULTS AND DISCUSSION Performance of the Present Axial Detection Scheme/ Application to Inert Solutes. The refractive index of Teflon AF2400 is 1.29, which is slightly lower than that of water (n ) 1.33), and thus, simple a LCW can be designed by filling the Teflon AF-2400 tube with aqueous running buffer.25-29 Stable solutes were first examined to assess the separation and detection performance of the present scheme. The inner diameter of the capillary used in this study is larger than that of a capillary usually used for CE experiments, and thus, the concentration of the running buffer should be kept as low as possible to minimize an electrical current; the cross-sectional area of the present capillary is ∼25 times as large as that of 50-µm-i.d. capillary. The concentration of solute was also kept low enough not to contribute to the conductivity of the sample zone. Figure 2A shows a change in the light intensity transmitted through the Teflon AF capillary, into which Ni2+(PAR)2 complex was injected as an inert sample. Under the studied condition (pH 8.38), both the complex and PAR itself are negatively charged, but eluted from the cathodic end of the capillary. The electrophoretic mobility measured with the usual CE experiments with terminal detection was -2.41 × 10-8 and -2.08 × 10-8 m2 V-1 s-1 for Ni2+(PAR)2 and PAR, respectively, indicating that the latter first leaves the capillary, and the former follows. A two-step increase in the light intensity thus results, and the signal becomes constant after all of compounds absorbing the probe light are eluted out of the capillary. Since this electropherogram is an integrated one, the differentiation of the transmitted light intensity gives a usual electropherogram as depicted in Figure 2B. The peaks of PAR and Ni2+(PAR)2 appear at 615 and 710 s, respectively; these times are shorter than those predicted from their mobilities, 1420 (for PAR) and 1640 s (for Ni2+(PAR)2). This strongly implies the presence of a flow toward the cathode, the velocity of which was estimated to be ∼3 × 10-4 ms-1 under this condition. This flow is predominantly caused by electroosmosis and partly by a small pressure difference between the inlet and outlet of the
Figure 4. Decomposition of Cd2+(PAR)2 induced by electrophoretic separation. (1) Injected sample (no applied voltage). (2) Immediately after the application of electrophoretic voltage. Equation 5 cannot be applied. (3) Excess PAR is completely separated from the Cd2+(PAR)2 zone. The decomposition has started to occur at any point in the Cd2+(PAR)2 zone. An absorbance change obeys eq 5. Figure 3. Change in transmitted light intensity during electrophoresis of Cd2+(PAR)2 complex. Running solution, 1 mM NaH2PO4 + Na2B4O7 (pH 8.38). Sample, 10 µM Cd2+ and 0.5 mM PAR. Length of Teflon AF-2400 tube, 25.5 cm. Applied voltage, 9 kV.
capillary as mentioned in the Experimental Section. As noted above, attention must be paid to pH changes in the running buffer during a CE operation. This effect may severely affect the performance and reliability of the present method, because the buffer concentration is quite low, to reduce electric currents. However, no signal change was detected before the elution of solutes; if pH were varied, the light intensity would also be changed because the spectrum of PAR was sensitive to a pH change. Thus, a change in the solution pH was sufficiently reduced by separating electrodes from buffer reservoirs (see Figure 1). The length of the sample zone was estimated to be 2.5 mm on the basis of the molar absorptivity of Ni2+(PAR)2; this length is consistent with that estimated from the injection by siphoning (5cm height for 2-3 s). If the axial diffusion were solely responsible for the zone broadening, the peak width should be calculated by the relation, x2 ) 2Dt, where x and D are the average displacement and the diffusion coefficient of a solute. In this case, the peak width of a solute zone just before it leaves the capillary is ∼4-5 mm, corresponding to ∼8-10 s. However, the actual peak width is ∼70-100 s, which is several times as large as that estimated by assuming axial diffusion. This must be due to use of a relatively large bore capillary and concomitant heat generation. Decomposition of a Labile Complex. Cd2+(PAR)2 complex was examined as a labile complex, and the kinetics of its decomposition was studied by the present method. This solute was studied in the kinetic mode CE and known to be decomposed during a usual CE separation process.11-13 The labile nature of this complex was confirmed by its injection into the running buffer not containing PAR; the migration of the complex was not detected by a terminal spectrometric detector. However, a use of the running buffer containing PAR gave two discrete peaks; the mobility of Cd2+(PAR)2 complex was estimated to be -2.67 × 10-8 m2 V-1 s-1 from this experiment. This solute was introduced into the LCW electrophoretic system, and its decomposition during the electrophoretic process was monitored with the axially introduced probe light. A result is shown in Figure 3. The entire feature of changes in the light intensity is different from that for Ni2+(PAR)2 illustrated in Figure 2; for Cd2+(PAR)2, the light intensity starts to increase immediately after the application of electrophoretic voltage, reaches plateau, again increases, and
finally becomes constant. The second increase in the transmitted light intensity is due to the elution of solutes; since, as shown by the usual CE experiments, Cd2+(PAR)2 is decomposed during separation, the elution of PAR should be responsible for the second increase in the transmitted light intensity. In contrast, the first change in the light intensity should have an origin different from the second one because this immediate increase was not seen for an inert complex such as Ni2+(PAR)2. The first increase in the light intensity must be caused by the electrophoretic decomposition of Cd2+(PAR)2, which should be a first-order reaction: k
Cd2+(PAR)2 98 Cd2+ + 2PAR
(1)
where k denotes the first-order kinetic constant of the reaction. The 1:2 complex may be dissociated with two steps. However, in the present scheme, the complete dissociation represented by eq 1 is monitored as an apparent one-step reaction; in other words, we cannot distinguish the first step from the second one of the dissociation process. This dissociation reaction will start when the zone of Cd2+(PAR)2 is electrophoretically separated from that of excess PAR as schematically illustrated in Figure 4. At a given small segment in the Cd2+(PAR)2 zone, from which free PAR has been electrophoretically removed, the reaction can be described by the following usual first-order rate equation:
C ) C0 exp(-kt′)
(2)
where C0 is the initial concentration of Cd2+(PAR)2 and t′ is the reaction time at a given part of the sample segment; it should be noted that t′ is different from t, which is the time elapsing after the electrophoretic separation has started. The point where the dissociation starts to occur is moved along the Cd2+(PAR)2 zone at the rate corresponding to the difference in the electrophoretic velocity between the Cd2+(PAR)2 and PAR (∆µ ) 5.9 × 10-9 m2 V-1 s-1). After these two solute zones are completely separated, the dissociation proceeds in the entire Cd2+(PAR)2 zone at rates depending on the time when the reaction started at a given point in a sample zone. For 9 kV applied voltages, it takes 12 s before this situation is reached, when the sample zone width is 2.5 mm. We can see this induction part in Figure 3 for the initial 10-20 s. After the dissociation occurs at the entire part of the solute zone Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
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(t >20 s in Figure 3), the absorbance coming from the Cd2+(PAR)2 complex can be written as
ACd-PAR ) Cl Cl )
∫
L
0
C exp(-k(t - t′)) dx )
∫
L
0
( ( vx)) dx
C exp -k t -
(3) where and C are the molar absorptivity and initial concentration of the Cd2+-PAR complex, respectively, v is a difference in the electrophoreteic velocity between Cd2+(PAR)2 and PAR, L is the length of the initial sample zone, and l is the time-dependent light path length. Beer’s law is well obeyed in the weakly guiding optical fibers used in the present work.31 At the detection wavelength, PAR also contributes to the overall absorbance.
APAR ) ′C′(L - l) + ′C′L
(4)
where ′, C′, and C′′ are the molar absoptivity of PAR, the concentration of PAR produced by the dissociation of the complex, and its excess concentration originally contained in the injected sample, respectively. Thus, the total absorbance change with time can be described by the following equation
Atotal ) ACd-PAR + APAR )
(C - ′C′)v L exp k - 1 exp( -kt) + k v ′(C′ + C′′)L (5)
( ( ) )
Thus, the change in measured absorbance with time obeys a simple exponential decay, and the first-order kinetic constant can be determined by a curve-fitting. A result of a curve-fitting is shown in Figure 5. The first-order kinetics constant, k, has thus been determined at 0.0226 s-1 (σ ) 0.004). The same experiments were also attempted with different voltages, e.g., with 6 kV, k ) 0.0233 s-1 (σ ) 0.006), which agrees well with that determined with a different applied voltage. It should be noted that the dissociation of Cd2+-PAR cannot be evaluated by other methods. Therefore, the reliability of the present scheme cannot directly be assessed by comparing this result with that obtained with an off-line measurement. The kinetics of the oxidation of Fe2+ in an acetate buffer was tested to confirm the (31) Dallas, T.; Dasgupta, P. K. Trends Anal. Chem. 2004, 23, 385, and references therein. (32) Abe, S.; Saito, T.; Suda, M. Anal. Chim. Acta 1986, 181, 203. (33) Mochizuki, K.; Imamura, T.; Ito, T.; Fujimoto, M. Bull. Chem. Soc. Jpn. 1978, 51, 1743. (34) Rossi, A. V.; Tubino, M. Ecletica Quim. 2003, 28, 55. (35) Funada, R.; Imamura, T.; Fujimoto, M. Bull. Chem. Soc. Jpn. 1979, 52, 1535. (36) Gupta, N.; Naik, R. M.; Nigam, P. C. Inorg. Chim. Acta 1989, 160, 103. (37) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476. (38) Plenert, M. L.; Shear, J. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3853.
4712 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
Figure 5. Absorbance change during the decomposition of Cd2+(PAR)2. Dots, experimental points derived from the data shown in Figure 3. Equation 5 was fitted to the experimental points.
validity of the present method. This oxidation is very slow, but can easily be detected in the presence of tiron (4,5-dihydroxy1,3-benzenedisulfonic acid), which forms a colored complex with the oxidation product, Fe3+. This reaction was utilized for kinetic determination of Fe2+ and Fe3+.32 Electrophoretic migration of Fe2+ injected into the acetate buffer containing tiron causes the oxidation of Fe2+, which can be followed by the axial detection scheme. Off-line measurements gave the pseudo-first-order reaction rate, ∼0.0009 s-1, which almost agrees with that determined by the present method under the same condition (k ) 0.0012 s-1). A number of kinetic data are available for the formation of PAR complexes; e.g. Co2+-PAR, Co3+-PAR,33 Cu2+-PAR, Zn2+-PAR,34 and Tl3+-PAR35 have been investigated with a stopped-flow technique in detail. In contrast, though the Cd2+(PAR)2 complexation has been widely utilized for the determination of Cd2+, kinetic studies of this complex have been very few. This complexation is so fast that the time change cannot be followed by the usual spectrophotometry. Gupta et al.36 investigated the dissociation of the Cd2+(PAR)2 complex in the presence of cyanide and suggested that the dissociation kinetic constant of this complex is smaller than 0.4 s-1. Although this cannot be directly compared with the corresponding value reported above, those evaluated by different methods are almost consistent. The present scheme is applicable to the reactions of k < 0.1 s-1, but could not probe faster reactions without modifications. However, its applicability can be extended if LCW is constructed in narrower separation channels. Extremely fast electrophoresis has recently been reported, in which separation within submilliseconds or some tens of microseconds has been realized with very narrow and short capillaries.37,38 If the present concept is applicable to these systems, reactions with k ∼ 103-106 are kinetically studied. Thus, CE can be a new tool for kinetic research by the appropriate combination with LCW. Received for review January 26, 2006. Accepted April 17, 2006. AC060175C
