Online Sample Preconcentration in Capillary Electrophoresis using

Aug 1, 2008 - Online Sample Preconcentration in Capillary Electrophoresis using Analyte Focusing by Micelle Collapse. Joselito P. ... Phone: 613 6226 ...
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Anal. Chem. 2008, 80, 6824–6829

Online Sample Preconcentration in Capillary Electrophoresis using Analyte Focusing by Micelle Collapse Joselito P. Quirino* and Paul R. Haddad Australian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Tasmania 7001, Australia A dimension for online sample preconcentration in capillary electrophoresis (CE) without modification of current CE commercial instrumentation is introduced. The focusing mechanism is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. More than 2 orders of magnitude improvement in detection sensitivity for model steroidal compounds using sodium dodecyl sulfate micelles as carrier is demonstrated. The capture, accumulation, transport, and release of molecules with carriers are highly important processes with vast practical applications.1-3 These processes often involve surfactant micelles and when this is the case they can be conveniently studied using micellar electrokinetic chromatography (MEKC), an analytical technique that uses micelles in solution as carriers for the separation of compounds in narrow channels under an applied electric field.4,5 Micelles are popularly known to solubilize material into a hydrophilic aqueous phase, although some have utilized the chemistry of micelles to also transport and release molecules into specific sites.6-8 Introduction of new mechanisms on the micellar transport, release, and accumulation of molecules will transform the use of these carrier-based processes in chemistry and related fields. Introduced by Terabe in 1984, MEKC is an important scientific tool that separates molecular analytes by a combination of electrophoretic and chromatographic effects. For example, sodium dodecylsulfate (SDS) present at a concentration above the critical micelle concentration (cmc) in a suitable electrolyte is used to form micelles that act as a hydrophobic pseudostationary phase with which the analytes interact in a manner analogous to the stationary phase material (e.g., modified silica) used in liquid * To whom correspondence should be addressed. Fax: 613 6226 2858. Phone: 613 6226 2163. E-mail: [email protected]. (1) Kostyniak, P. J.; Clarkson, T. W. Fundam. Appl. Toxicol. 1981, 1, 376. (2) Wu, G. Y.; Zhan, P.; Sze, L. L.; Rosenberg, A. R.; Wu, C. H. J. Biol. Chem. 1994, 269, 11542. (3) Marrelli, M. T.; Li, C.; Rasgon, J.; Jacobs-Lorena, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5580. (4) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (5) Palmer, C. P.; Terabe, S. Anal. Chem. 1997, 69, 1852. (6) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615. (7) Bae, Y.; Jang, W.-D.; Nishiyama, N.; Fukushima, S.; Kataoka, K. Mol. BioSyst. 2005, 1, 242. (8) Zhu, H.; McShane, M. J. J. Am. Chem. Soc. 2005, 127, 13448.

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chromatography.4 An inline UV absorbance detector is commonly utilized to visualize the analytes in MEKC. MEKC and other electrophoretic and electrochromatographic techniques are nowadays used frequently as separation modes in capillaries and in analytical microfluidic devices, but detection in the narrow channels of these devices is often difficult and the use of an online preconcentration technique is often required in order to ensure a suitable detection signal. For neutral and charged analytes, common techniques for online preconcentration are field-enhanced/ amplified sample stacking, sweeping, self-focusing with photopolymerized porous monoliths, and solvent gradient effects. Alternative preconcentration methods involve transient isotachophoresis, isoelectric focusing, and dynamic pH junction focusing, but these can be used for charged analytes only.9-14 By manipulation of the solution chemistry in MEKC, we here demonstrate the striking transport, release, and accumulation of neutral molecules from the hydrophobic core of an anionic micelle into a hydrophilic (liquid) phase. This is opposite to the wellknown “like-dissolves-like” approach in which hydrophobic molecules may be concentrated into a hydrophobic matrix.15,16 In the present work, the sample molecules are transported inside micelles under an applied electric field in a micellar electrolyte solution containing an anionic surfactant and an additional anion having high electrophoretic mobility. With an increase in the concentration of added electrolyte salt in the sample, the surfactant micelles are continuously diluted and collapsed into a liquid phase zone, thereby releasing and accumulating the transported molecules (see Figure 1). Micellar electrokinetic dilution, where the concentration of the micelle drops below its cmc, occurs through movement of the micelle and the electrolyte anion into a dilution zone that follows the Kohlrausch regulating function (KRF) for electrophoretic systems.17-19 This process, which we call analyte focusing by micelle collapse (AFMC), is a new dimension for (9) Liu, Z.; Sam, P.; Sirimanne, S. R.; McClure, P. C.; Grainger, J.; Patterson, D. G. J. Chromatogr., A 1994, 673, 125. (10) Quirino, J. P.; Terabe, S. Science 1998, 282, 465. (11) Simpson, S. L.; Quirino, J. P.; Terabe, S. J. Chromatogr., A 2008, 1184, 504. (12) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023. (13) Britz-McKibbin, P.; Bebault, G. M.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729. (14) Quirino, J. P.; Dulay, M. T.; Zare, R. N. Anal. Chem. 2001, 73, 5557. (15) Li, G.; Fudickar, W.; Skupin, M.; Klyszcz, A.; Draeger, C.; Lauer, M.; Fuhrhop, J.-H. Angew. Chem., Int. Ed. 2002, 41, 1828. (16) Horvath, I. T.; Rabai, J. Science 1994, 266, 72. (17) Kohlrausch, F. Ann. Phys. (Leipzig, Ger.) 1897, 62, 209. (18) Dismukes, E. B.; Alberty, R. A. J. Am. Chem. Soc. 1954, 76, 191. 10.1021/ac801258r CCC: $40.75  2008 American Chemical Society Published on Web 08/01/2008

Figure 1. Transport, release, and accumulation of analytes in electrokinetic chromatography by micelle collapse in a dilution zone.

online sample preconcentration and control in MEKC that is limited by chemical diffusion and dispersion caused by local electroosmotic flow (EOF) velocity mismatch.20,21 This sample control is also a nanopreparative step compatible with analysis in microfluidic devices and mass spectrometry.22-26 Alternatively, focused sample may be collected in a small vessel for further reactions or manipulations. MATERIALS AND METHODS All experiments were performed on an Agilent HPCE system (Waldbronn, Germany) with an inline UV detector (wavelength set at 254 nm). Polyimide-coated untreated fused-silica capillaries were obtained from Polymicro Technologies (Phoenix, Arizona) (50 µm i.d.). These capillaries were conditioned appropriately and thermostatted at 20 °C in all experiments. All reagents were analytical grade and water was obtained from a Milli-Q system. All samples were obtained from Sigma with purity >99%. All solutions were passed through a 0.45 µm filter prior to use. RESULTS AND DISCUSSION Theory of Micellar Electrokinetic Dilution and Micelle Collapse. In electrophoresis with strong electrolytes, the Kohlrausch regulating function of a zone (x) is given by eq 1.19 The total flux of ions in and out of x is conserved by a value equal to the KRF. zi is the charge of the ion i, ci(x,t), is the concentration of i at zone x at time t, and µi is the electrophoretic mobility of i. N

KRF(x) )

∑ i)1

|zi|ci(x, t) µi

(1)

Figure 2 shows the first system used in this study. Under initial conditions (Figure 2a) the capillary contains an electrolyte zone (x1) containing a high electrophoretic mobility anionic component (19) Hruska, V.; Gas, B. Electrophoresis 2007, 28, 3. (20) Muijselaar, P. M.; van Straten, M. A.;.; Claessens, H. A.; Cramers, C. A. J. Chromatogr., A 1997, 766, 187. (21) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991, 63, 2042.

(designated hma-), such as phosphate. A sample zone (x2) containing the same electrolyte component, an anionic micelleforming agent (mc-) such as SDS, and the sample molecules (s) is then injected into the capillary. x1 and x2 are comprised of solution 1 and solution 2, respectively. xb is the boundary zone which separates the two zones. The zone closest to xb in solution 1 is called the micellar dilution zone (MDZ). The concentrations of each species in the various zones are depicted in the figure. When an electric field is applied (Figure 2b), ions move in the direction of the anode and cathode, depending on their charge. For simplicity, the effect of the cationic counterion present in the system is neglected. At the MDZ or zone directly at the right of xb in Figure 2b, the anions from solution 2 (mc- and hma-) progressively replace the anions in solution 1 (hma-). Initially, the MDZ is completely filled by hma- from solution 2, but mc- moves into this zone and its concentration increases from an initial value of zero to a value cmc(xMDZ,t) approximated by eq 2. Equation 2 was derived on the assumption that the anions (mc- and hma-) in x2 will be diluted by the same factor (ratio of KRF in solution 1 and solution 2) at the MDZ.18

cmc(xMDZ, t) ) cmc(x2, 0)

KRF(x1) KRF(x2)

(2)

From eq 2, when cmc(xMDZ,t) is less than the cmc of the surfactant, the micelle collapses with the subsequent release of any analyte molecules bound hydrophobically to the interior of the micelle. The continued application of the electric field causes more micelles from solution 2 to collapse and more neutral analytes to be transported to the boundary. The AFMC phenom(22) Sera, Y.; Matsubara, N.; Otsuka, K.; Terabe, S. Electrophoresis 2001, 22, 3509. (23) Belder, D.; Ludwig, M.; Wang, L.-W.; Reetz, M. T. Angew. Chem., Int. Ed. 2006, 45, 2463. (24) Liu, Y.; Foote, R. S.; Jacobson, S. C.; Ramsey, J. M. Lab Chip 2005, 5, 457. (25) Przybylski, M.; Glocker, M. O. Angew. Chem., Int. Ed. 1996, 35, 806. (26) Jo, K.; Heien, M. L.; Thompson, L. B.; Zhong, M.; Nuzzo, R. G.; Sweedler, J. V. Lab Chip 2007, 7, 1454.

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Figure 2. Illustration showing the mechanisms of electrokinetic micellar dilution and collapse with subsequent focusing of analyte zones. The left panel (a) shows the initial conditions (t ) 0) where the electroosmotic flow is neglected for simplicity. The y-axis is the concentration and x-axis is the length of the channel (L). The cathode is at the left side while the anode close to a detector is at the right side. The x1 zone is formed from injection of solution 1 that contains the high mobility anion (hma-). The x2 zone is formed from injection of solution 2 that contains the hma-, the micelle forming agent (mc-), and the analyte sample (s). The xb boundary separates the two solutions zones and is stationary in the absence of electroosmotic flow. The concentration of hma- in the sample solution (solution 2) is adjusted to a high value, such that the concentration of the micelle forming agent that will penetrate the x1 zone during application of voltage will be lower than the cmc. The concentration of the micelle forming agent in the sample solution is above the cmc, where micelles are formed. With the application of voltage (b), the micelles from x2 zone move in the direction of x1. The hma- being faster than the mc-, reaches the xb first. The zone in x1 closest to the xb (micellar dilution zone or MDZ) is thus initially filled by hma-. The mc- catches up and shares the KRF in the MDZ with the hma-. In effect, concentration gradients of the two anions are formed from the boundary (right-hand side of part b). A steady state is reached where the concentration of both anions that fill the MDZ is constant. If the concentration of the micelle forming agent that enters the MDZ is lower than the cmc, the micelles will collapse, and in the process release the analyte molecules into this liquid phase zone. The continuous electrophoresis of the micelle to the other zone allows more molecules to be released at the liquid phase MDZ causing the accumulation and concentration of analytes at this zone. Chemical diffusion and other effects cause the broadening of the focused analyte zone.

enon is therefore the transport, release, and accumulation of bound molecules using a micellar phase that collapses as it dilutes to a concentration below its cmc into an aqueous zone. Micellar electrokinetic dilution causing micelle collapse is maintained at the boundary unless the boundary is pushed out of the capillary, for example, by electroosmosotic flow (EOF) or external pressure. This boundary can also be disrupted by the dispersive effects of EOF mismatch. The caption in Figure 2 also gives a discussion of the processes involved in AFMC. Experimental Application of AFMC. Figure 3 shows the experimental verification of AFMC. The capillary was conditioned with solution 1 (micellar electrokinetic dilution solution) and then vials containing solution 1 and solution 2 (sample solution) were placed at the anodic and cathodic ends of the capillary, respectively. UV detection was performed at the anodic end. A voltage of 5 kV was applied across the capillary with 50 mbar positive pressure toward the anode being used to move the boundary between zones x1 and x2 to the detector. In general, a steady baseline was initially observed (due to solution 1), followed by detection of the MDZ, the boundary, and then a higher signal from solution 2 due to the analytes in the sample. At the correct proportions of high mobility anion (i.e., red electrochromatogram, 90 mM phosphate in solution 2 and 40 mM in solution 1) and micelle (i.e., 5 mM SDS) in the solutions used, the micelles from the sample solution (zone x2) were collapsed at the MDZ accompanied by the release and accumulation of the test analytes. MDZ is the zone adjacent to the boundary (xb) found in solution 1. This focusing is evidenced by the greatly increased detector signal at the MDZ. 6826

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The approximated cmc of SDS in the systems we used is 3 mM (no analyte retention was observed with micellar buffers containing less than 3 mM SDS in MEKC) while others have reported values below 3 mM.27 For the red electrochromatogram, the resulting concentration of SDS in the MDZ is predicted to be below the cmc. Altering solution 2 to contain 60 (blue electrochromatogram) and 30 mM (green electrochromatogram) sodium phosphate (pH 1.7) produced SDS concentrations of approximately 3.4 and 6.5 mM in the MDZ, respectively. With 30 mM (green) phosphate in the sample, stacked micelles of >5 mM SDS were present in the MDZ at detection, as evidenced by the small hump at the vicinity of the xb and this is consistent with previous findings.28No analyte accumulation was observed because the analytes were not released but were retained and transported by the micelle as shown by the steady detector response at the left of the xb. With 60 mM (blue) sodium phosphate at pH 1.7 in the sample, micelles of SDS persisted in the MDZ but at a lower concentration than that of the sample zone (5 mM). Dilution of the analytes (lower signal) therefore occurred in the MDZ. This process may also be utilized as a source for sample control, to deliver lower amounts of material, but micelles are delivered as well. With 90 mM sodium phosphate at pH 1.7 in the sample, the concentration of SDS in the MDZ was less than the cmc, so that the micelles collapsed and the analyte was focused. This focusing is evidenced by the greatly increased detector signal at the MDZ. A dispersive effect produced by the local electroosmotic flow (27) Fuguet, E.; Rafols, C.; Roses, M.; Bosch, E. Anal. Chim. Acta 2005, 548, 95. (28) Giordano, B. C.; Newman, C. I. D.; Federowicz, P. M.; Collins, G. E.; Burgi, D. S. Anal. Chem. 2007, 79, 6287.

Figure 3. Experimental verification of AFMC. Solution 1 (x1) is 40 mM sodium phosphate buffer, pH 1.7 while solution 2 (x2) is 5 mM SDS in 30, 60, or 90 mM sodium phosphate buffer, pH 1.7 with 0.9 µg/mL prednisolone. E is electric field strength while P is pressure. Capillary length is 28.5 cm total (8.5 cm to the detector). For the red electrochromatogram, MDZ is the micellar dilution zone where the micelles from x2 are diluted and collapsed. The collapse of the micelles causes the previously bound analytes to be released at the MDZ. Continued collapse of the micelle and release of the bound analytes cause the accumulation of the analytes forming a more concentrated zone of sample at the MDZ. More explanation is found in the text.

mismatch due to difference in conductivity of the solutions inside the capillary is present. This effect may not be significant since the difference in conductivity between the sample and separation solution in the AFMC experiments performed is less than 10.21 The effect of solution conductivity in AFMC will be discussed elsewhere. It is also possible to perform AFMC using only the surfactant molecules (no electrolyte salt) in solution 1 (background solution) and solution 2 (sample matrix). Using 3 mM SDS as solution 1 and 10 mM SDS as solution 2, we observed focusing of hydrocortisone similar to that found in Figure 3 (red electrochromatogram). Note that the cmc of SDS in water is around 8 mM. In the presence of an electric field, the SDS micelles from solution 2 are diluted to a concentration below the cmc (∼3 mM) at a zone (in solution 1) directly adjacent to the boundary between solution 1 (3 mM SDS) and solution 2 (10 mM SDS). The phenomenon of micelle collapse in this case is more straightforward since the surfactant molecules from solution 2 simply replace the surfactant molecules in solution 1. Surfactant micelles from solution 2 collapse since the surfactant concentration in solution 1 is lower than the cmc. No focusing effect was observed when solution 1 and solution 2 contains the same concentration of SDS (3 or 10 mM). Figure 4 shows the control of the sample preconcentration process by varying the applied potential. The preconcentration factor could be increased with an increase in the electric field strength (V/cm), as shown in Figure 4 (left), or with an increase in voltage application time at a given electric field strength (179

Figure 4. Controlling the amounts of sample concentrated by AFMC. For electric field strength experiments (left), the conditions are similar to Figure 3 (red) except the voltage was stopped at 2 min and pressure was continued to visualize the focused zones. Electric field strengths (a) 0, (b) 71, (c) 179, (d) 357, (e) 714, and (f) 1071 V/cm. Note that at 0 V/cm, no focused zone (/) was formed. Electrochromatograms are drawn on the same scale. For voltage application time experiments (right), detection of the AFMC zone at 2.5 min (g), the conditions are similar to that found in Figure 3 (red). For detection of AFMC zone at 10.2 min (h), the longer effective length was used (20 cm to the detector) for injection of sample solution. Electrochromatograms are drawn on the same scale. s is the focused sample zone.

V/cm), as shown in Figure 4 (right). At least an order of magnitude improvement in concentration could be obtained using both approaches. The retention factor (k) of the analyte molecules in AFMC drops to zero at the point of micellar collapse. The analytical concentration of the enriched sample zone may therefore theoretically increase progressively with continuation of the AFMC process. However, this increase is hindered by diffusion, since the diffusivity of the analyte molecule is at least 10 times greater outside the micelle than within the micelle, such that diffusion of the analyte away from the boundary zone will ultimately limit the preconcentration factor that can be achieved within a given period of time.20 The convective effects caused by changes in local EOF at the boundary between solution 1 and 2 may also affect the focusing of zones in AFMC, although this effect will be minimal when the conductivity difference between solution 1 and 2 is not greater than a factor of 10.21 In addition, for very hydrophobic analytes, surfactant monomer-analyte interaction may occur and cause some broadening of focused zones. AFMC Preconcentration Coupled with MEKC Separation. The combination of sample enrichment by AFMC and separation by MEKC is illustrated in Figure 5 using steroids as analytes. The stages of AFMC in MEKC are shown schematically in Figure 5a and the resultant electrochromatogram (see Figure 5b,2, bottom electrochromatogram) can be compared to that obtained with a direct injection and MEKC separation of a 100-fold more concentrated sample (see Figure 5b,1, top electrochromatogram). Figure 5a,1 is the starting situation, in which the capillary is conditioned with solution 1 (light blue) followed by pressure injection of the sample solution (solution 2, gray zone). Solution Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 5. (a) AFMC MEKC model (explanation in text), (b) online sample preconcentration of steroids by AFMC MEKC. BGS (solution 1): 100 mM SDS in 2 mM sodium phosphate buffer, pH 10 and 20% methanol. Sample matrix: separation solution (b1) and 3 mM SDS in 80 mM sodium phosphate, pH 10 (solution 2) (b2). Concentration of steroids: 0.7-1.2 mg/mL (b1) and 7-12 µg/mL (b2). Capillary length is 50.5 cm total (42 cm to the detector). Injection length: 0.15 cm (50 mbar for 2 s) (b1) and 8.25 cm (50 mbar for 106 s) (b2). Electric field: 495 V/cm. Peak identity: cortisone (c), hydrocortisone (h), and prednisolone (p).

1 fills vials at both ends of the capillary. Figure 5a,2 starts the application of voltage where the analytes are focused (dark gray region) at the anodic end of the sample band due to migration of micelles to the MDZ and subsequent collapse. The EOF is toward the cathode, so all zones are carried toward the cathode and the detector. Micelles from solution 1 move progressively into solution 2 (dark blue region) and ultimately reach the MDZ containing the enriched sample zone (Figure 5a,3). The concentration of SDS at the MDZ do not fall below the cmc and will be similar to that present originally in solution 1. Separation of the focused sample bands then occurs by MEKC, as shown in Figure 5a,4 (light blue region with separated focused sample given by dark shaded bands). Figure 5a,4 also shows the subsequent transport of the focused bands to the detector according to conventional MEKC principles. Note that solution 1 introduced from the inlet end does not actively participate in the focusing process and is therefore not shown in Figure 5a. Figure 5b shows 160-200-fold increases in peak heights for three test steroids which have been focused by AFMC and then separated by MEKC. These increases were calculated by dividing 6828

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the peak height obtained with AFMC by that obtained for conventional injection, after correction for the dilution factor. Under the conditions used, solution 1 contained 100 mM SDS in 2 mM sodium phosphate (pH 10)/20% methanol and solution 2 contained 3 mM SDS in 80 mM sodium phosphate (pH 10). The % RSD values (n ) 3) for retention time and corrected peak areas for the three analytes ranged from 5.0-8.1 and 2.2-4.4, respectively. AFMC relies on the micelles to transport, release, and accumulate analytes into the MDZ. The transport of analytes with high affinity to the micelle, such as those found in Figure 5, is therefore very efficient. However, the magnitude of the EOF limits the length of the sample plug injected because at high EOF the focused zone may have insufficient time to separate before reaching the detector. The effective separation length in AFMC (Figure 5b2) was shortened by the injection plug length and this caused the focused analytes to be detected faster compared to the conventional injection method in Figure 5a1. A longer capillary was then used in Figure 5b compared to Figures 3 or 4 to allow separation of AFMC concentrated zones. The enhancement factor differs between analytes because the rate of transport to the boundary zone is dependent on the k value of the analyte in the micelle and also on the rate of diffusion of the analyte after micelle collapse. For an injection length greater than that shown in Figure 5b (i.e., 13.7 cm), increases in peak height were from 470 to 730fold, but resolution of the last two peaks was compromised as the peaks were not given enough time to separate (all peaks eluted within 8 min). Earlier detection of focused zones will minimize the broadening effects of diffusion and similar behavior has been observed in sweeping.22 With the 13.7 cm injection length, the estimated limits of detection (S/N ) 3) were in the range 0.003-0.008 µg/mL. Up to 100-fold improvements in peak heights were also obtained using AFMC for other neutral analytes (alkylphenyl ketones and dialkyl phthalates). Preconcentration of similar compounds by sweeping was better (sometimes greater >5000-fold) because micelles penetrate the sample zone and therefore significantly reduce the diffusivity of the analyte molecules.10 Moreover, in sweeping, the sample matrix can be prepared with similar conductivity to the separation solution, thus removing the dispersive effects of local EOF mismatch. AFMC and sweeping have opposite mechanisms of action: the micelles leave the sample zone in AFMC and in sweeping they penetrate the sample zone. AFMC also works for analytes with charge and this will be described elsewhere. CONCLUSION Two fundamental electrokinetic phenomena, micellar electrokinetic dilution and AFMC, in MEKC have been presented. These phenomena are not only present in microscale electrophoretic separations but also in various systems where carriers, high mobility anions, and electric fields are involved. For example in iontophoretic drug delivery, hydrophobic molecules were found to accumulate in the polar intracellular chamber.29 This unusual accumulation may be explained by the dilution of cell membrane components, which are historically mimicked by micelles, causing release of the hydrophobic molecules that have penetrated the cell membrane by diffusion. We will study the use of these (29) Turner, N. G.; Guy, R. H. J. Pharm. Sci. 1997, 86, 1385.

phenomena to intracellular molecular and iontophoretic drug delivery. It is possible to apply these approaches for the microscale manipulation of products obtained from micellar nanocontainers or nanoreactors, to study, for example, reaction kinetics or/and dynamics. AFMC is an attractive approach to online preconcentration and nanopreparative preconcentration of both charged and neutral compounds in capillary zone electrophoresis, MEKC, and capillary electrochromatography where carriers and high mobility anions are present in the sample matrix. These matrixes are typically found in real world biological samples. There is also no need to modify current commercial instrumentation, making AFMC easily adoptable to other laboratories. We will apply AFMC in microfluidic devices and detection with mass spectrometry. Moreover, the controlled release of material from carriers is a

hurdle in various applications, and our approach provides a fundamental understanding on how to release and accumulate molecules bound in micellar systems. We expect this work to be a springboard to solving problems in sample transport, release, and accumulation as well as in chemical and biological analysis. ACKNOWLEDGMENT This work is dedicated to Prof. Shigeru Terabe. The authors thank Prof. Shigeru Terabe, Prof. Richard Zare, and Prof. C. P. Palmer for their comments. Received for review June 23, 2008. Accepted July 23, 2008. AC801258R

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