Anal. Chem. 2007, 79, 5082-5086
Magnetically Immobilized Beds for Capillary Electrochromatography Yucong Wang, Zhichao Zhang, Lei Zhang, Feng Li, Lei Chen, and Qian-Hong Wan*
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
Capillary electrochromatography (CEC) is a hybrid separation technique that couples the high separation efficiency of capillary electrophoresis with the superior selectivity of high-performance liquid chromatography.1-3 Despite the fact that CEC is recognized as of tremendous potential in the pharmaceutical and biomedical fields, several technical problems have to be tackled before the technique enjoys general acceptance and finds widespread applications. One such problem is associated with column preparation.4 Conventional methods for preparation of particle-packed capillary columns include (i) slurry packing of reversed-phase silica into a fused-silica capillary with frits formed by thermal sintering to hold the packing material inside the capillary;5-8 (ii) pulling of a glass tube filled with packing material followed by in situ derivatization;9 (iii) in situ formation of a monolithic or continuous bed in a capillary.10-14 Each of these methods has advantages and limitations. For instance, with the slurry packing
technique, the stationary phase can be chosen from a wide variety of commercial HPLC packing materials and previous experience gathered in HPLC can be readily transferred to CEC. Nevertheless, it suffers from problems associated with frit fabrication. The frits produced by thermal sintering cause the nonuniformity of the packed bed that is liable for bubble formation during the operation. Pulling the glass tube at high temperature produces packed capillaries with high permeability. Without polymeric coatings, however, the glass columns are fragile and difficult to use. The monolithic columns hold great potential for CEC as they are free from the problems associated with particle packing and frit fabrication. Unlike the slurry packing method, however, the monolithic approach lacks the flexibility with regard to the selection of stationary phases. Clearly, there is still a need for developing alternative column preparation methods that are complementary to those currently available. We wish to report a magnetic field-assisted immobilization approach to fritless packed columns for CEC. The use of magnetic fields for confinement of particles has a long history of applications in chemical and biological engineering.15-18 A notable example in this area is the magnetically stabilized bed.19 A fluidized bed is formed by applying fluid drag forces on a mass of solid particles whereby the behavior of the mass of the fluidized bed corresponds to that of a liquid. Fluidized beds possess many desirable attributes in temperature control, heat transfer, and chemical reaction; they therefore have found widespread applications in numerous technological processes. One of the major problems associated with fluidized beds is that of bubble formation, frequently resulting in slugging, channeling, spouting, attrition, and pneumatic transport. The formation of bubbles and slugs in a fluidized bed may be eliminated by an external magnetic field due to the interaction of this field with fluidized ferromagnetic particles. In general, a uniform, time-steady magnetic field oriented parallel with the direction of fluid flow is used to produce a stably fluidized bed
* Corresponding author: (phone) +86-22-2740-3650; (fax) +86-22-2740-3650; (e-mail)
[email protected]. (1) Knox, J. H.; Boughtflower, R. Trends Anal. Chem. 2000, 19, 643-647. (2) Colon, L. A.; Burgos, G.; Maloney, T. D.; Cintron, J. M.; Rodrigues, R. L. Electrophoresis 2000, 21, 3965-3993. (3) Bartle, K. D.; Myers, P. J. Chromatogr., A 2001, 916, 3-23. (4) Fujimoto, C. Trends Anal. Chem. 1999, 18, 291-301. (5) Knox, J. H.; Grand, I. H. Chromatographia 1991, 32, 317-326. (6) Chen. J.-R.; Dulay, M. T.; Zare, R. N.; Svec, F.; Peters, E. Anal. Chem. 2000, 72, 1224-1227. (7) Chen, Y.; Gerhardt, G.; Cassidy, R. Anal. Chem. 2000, 72, 610-615. (8) Piraino, S. M.; Dorsey, J. G. Anal. Chem. 2003, 75, 4292-4296. (9) Knox, J. H.; Grant, I. H. Chromatographia 1987, 24, 135-143. (10) Ou, J.; Li, X.; Feng, S.; Dong, J.; Dong, X.; Kong, L.; Ye, M.; Zou, H. Anal. Chem. 2007, 79, 639-646.
(11) Hutchinson, J. P.; Zakaria, P.; Bowie, A. R.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2005, 77, 407-416. (12) Lammerhafer, M.; Svec, F.; Frechet, J. M. J.; Lindner, W. Anal. Chem. 2000, 72, 4623-4628. (13) Kato, M.; Sakai-Kato, K.; Matsumoto, N.; Toyo’oka, T. Anal. Chem. 2002, 74, 1915-1921. (14) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921-3926. (15) Tong, X.-D.; Sun, Y. Biotechnol. Prog. 2003, 19, 1721-1727. (16) Seibert, K. D.; Burns, M. A. Biotechnol. Prog. 1998, 14, 749-755. (17) Sornchanmni, T.; Atwater, J. E.; Akse, J. R.; Wheeler, R. R., Jr.; Jovanovic, G. N. Ind. Eng. Chem. Res. 2005, 44, 9199-9207. (18) Ozkara, S.; Akgol, S.; Canak, Y.; Denizli, A. Biotechnol. Prog. 2004, 20, 1169-1175. (19) Rosensweig, R. E. U.S. Patent 4,115,927, 1978.
Fritless packed beds comprised of magnetically responsive octadecylsilane bonded silica particles have been constructed for reversed-phase electrochromatography. The magnetic particles were immobilized in the capillary by applying an external magnetic field transverse to the direction of electroosmotic flow. Being subjected to the interplay of fluid dragging and magnetic forces, the initial loosely packed particle assembly was compacted into a uniform packing structure. The magnetically immobilized beds obtained were used as stationary phases for separation of neutral compounds, with retention behavior and column efficiency similar to those of slurry-packed columns. The results suggest that the magnetic attraction approach to fritless column packing may be used for construction of advanced chip-based chromatography, especially in complex architectures comprising curved and intersecting channels.
5082 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
10.1021/ac070288b CCC: $37.00
© 2007 American Chemical Society Published on Web 06/01/2007
over a wide range of fluid velocities. Our objective here is to illustrate a new approach for preparation of densely packed beds for chromatographic separations using an external magnetic field to counteract the fluid dragging forces exerting on the packing material. The strategy reported here involves placing, trapping, and packing the magnetically responsive packing material in a capillary column by a transverse magnetic field. The magnetic packing material is hydrodynamically pulled into the capillary and retained in a region where the magnets are located. The loose plug of the magnetic particles thus formed is compacted by an electroosmotic flow which drives the magnetic particles against the influence of the magnetic field. As a result, the interplay of the dragging and magnetic forces produces a dense packing structure required for high-efficiency chromatographic separations. Clearly, the packing materials useful in this approach differ from conventional ones in that they are capable of acting in response to the magnetic field applied. An earlier report from this laboratory described a novel procedure for preparation of magnetically responsive silica microspheres as adsorbents for purification of genomic DNA.20 This procedure was adapted in the present work for preparation of magnetic silica-based reversed-phase stationary phases, which have strong magnetic susceptibility, large surface area, and uniform particle size distribution. The packing materials thus prepared were used to generate magnetically immobilized beds, in which high-efficiency reversed-phase separations of neutral compounds were performed. EXPERIMENTAL SECTION Chemicals and Materials. All chemicals used for preparation of magnetic silica were of analytical grade unless otherwise specified. Silica sol (30% SiO2, 10-15 nm) was obtained from Guolian Chemical Co. (Jiangying, China). n-Octadecyltrichlorosilane (95%) was obtained from Acros Organics (Morris Plains, NJ). HPLC grade methanol (Concord Tech Co. Tianjin, China) and doubly distilled water were used to prepare the mobile phases. The fused-silica capillaries used in this work were purchased from Xinnuo Chromatographic Products (Handan, China). Synthesis and Characterization of Magnetic Bonded Phases. The synthesis of magnetic bonded phases involves preparation and modification of magnetic silica microspheres. The preparation of magnetic silica microspheres was previously described in detail.20 Briefly, a colloid suspension of hematite was obtained by dissolving sodium hydrogen carbonate powders into an aqueous solution of iron(III) chloride. The hematite sol was then mixed with silica sol in the desired proportion. To the mixed sol were added urea, formaldehyde (37%), and concentrated HNO3 with stirring until a pH of 2 was reached. The reaction was allowed to proceed overnight at ambient temperature. The resulting composite microspheres were collected, washed, dried, and then subjected to staged-heating treatments. The porous hematite/silica composite particles were further treated by hydrogen reduction to afford highly magnetizable silica particles. The magnetic porous silica obtained above was used as a support for n-octadecyl chemically bonded phases. A total of 5 g of the magnetic silica was dried overnight at 90 °C under vacuum (20) Zhang, Z.; Zhang, L.; Chen, L.; Chen, L.; Wan, Q.-H. Biotechnol. Prog. 2006, 22, 514-518.
and dispersed in a flask containing 50 mL of toluene over molecular sieve. A volume of 4 mL of n-octyltrichlorosilane was added to the magnetic silica dispersion together with 2 mL of triethylamine. The mixture was refluxed for about 18 h under nitrogen atmosphere, and the product was then filtered and washed sequentially with toluene, methylene chloride, ethanol, and acetone. The magnetic ODS particles were dried overnight at 60 °C under vacuum. The morphology and size of the magnetic ODS particles were examined by scanning electron microscopy (SEM, X-650, Hitachi, Japan). The magnetic properties of the particles were studied using a vibrating sample magnetometer (LDJ 9600-1, LDJ Electronics Inc., Troy, MI) operating at maximum magnetic field of 10 kOe. Preparation of Magnetically Immobilized Beds. Prior to installation, the polyimide coating of a 250 µm i.d. fused-silica capillary was removed at a small section (3 mm long) that was 10 cm away from the outlet to create a detection window for CEC experiments. The outlet of the capillary was placed in a vial containing 5% (w/v) magnetic ODS particles dispersed in a mobile phase consisting of 80% (v/v) methanol and 20% (v/v) 5 mM phosphate buffer (pH 8.0). At the other end of the capillary was attached a 1 mL hypodermic syringe. The slurry of the magnetic ODS particles was introduced into the capillary by gently withdrawing the plunger of the syringe. When the capillary was filled with a desired amount of the particles, two identical cylindrical neodymium-iron-boron magnets (8 mm thick × 10 mm bore) were placed on opposite sides of the capillary at a distance of 20 cm from the outlet. The inlet end of the capillary was then placed in a vial containing the mobile phase only, and a high voltage of typically 15 kV was applied across the capillary. The magnetic particles were carried through the capillary by the electroosmotic flow and trapped in a region where the cylindrical magnets were located. A second magnet assembly was placed on the other end of the packed bed to lock the particles in place. The packed bed was further conditioned by driving the mobile phase through the capillary at an applied voltage of 15 kV for periods of time ranging from 30 min to 1 h. The magnetically stabilized column was ready for sample injection when both the current reading and detection signal were stabilized. Reversed-Phase Capillary Electrochromatography. The CEC experiments were carried out on a TH2000 HPCE system (Tianhui Institute of Separation Sciences, Baoding, China) equipped with a UV absorbance detector, a pressure-actuated injector, and a BaseLine chromatographic data acquisition and analysis system (BaseLine Chromtech Research Center, Tianjin, China). The fused-silica capillary with a detection window was installed, and the filling and trapping of the magnetic ODS particles in the capillary were carried out as described above. The mobile phase employed was a 5 mM phosphate buffer (pH 8.0) containing various percentages of methanol. The samples were injected electrokinetically at the anodic end of the capillary and detected at a wavelength of 254 nm. Separations were performed at an applied voltage of 15 kV and at ambient temperature. RESULTS AND DISCUSSION Magnetic Bonded Phases. The magnetic silica-based packing material used in this work was synthesized by following a literature procedure. Figure 1 shows scanning electron micrographs (SEM) Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
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Figure 1. Scanning electron micrographs of magnetic silica microspheres: (a) 3.0 µm diameter and (b) 1.0 µm diameter.
Figure 3. (a) Photograph of fritless packed capillary column formed between magnet assemblies. (b) Magnified (160×) views of packed capillary sections (inlet end, middle, and outlet end). Figure 2. Room-temperature magnetization curves for magnetic silica microspheres: (a) 3.0 µm diameter and (b) 1.0 µm diameter.
of two preparations of magnetic ODS particles with particle diameters being about 3.0 and 1.0 µm, respectively. The particles are nearly spherical in shape and relatively uniform in particle size distribution. Figure 2 shows hysteresis loops of the magnetic particles registered at room temperature. The saturation magnetizations (Ms) are 8.367 and 17.57 emu/g, respectively, for 3.0 and 1.0 µm particles. These Ms values are comparable to those for commercial magnetic polystyrene microspheres which have typical values of less than 10 emu/g. The spherical particle shape and uniform particle size distribution are essential for obtaining a permeable and homogeneous packing structure and thus providing high column efficiency. The high magnetization of the particles is a prerequisite for formation of a stable bed under a 5084 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
magnetic field. The distinct properties of the magnetic ODS phases presented in this work suggest their potential utility in magnetically assisted separation systems. Magnetically Immobilized Beds. The ability to generate a fritless packed bed in a flow channel facilitates construction of capillary- or chip-based chromatographic separation systems. Figure 3 shows sections of capillaries where magnetic ODS particles were immobilized by magnet assemblies placed outside of the capillary. The formation of densely packed bed is believed to be associated with the influences of fluid dragging and magnetic attraction forces. When an electric field was applied across the capillary filled with magnetic particles, an electroosmotic flow is induced and passed through interparticle passages. Due to the viscous nature of the liquid, the electroosmotic flow transports
the magnetic particles toward the outlet of the capillary.21 The liquid flow passes through the region between the magnetic disks, but the magnetic particles are retained due to their interaction with the magnetic field. A dense packing structure results from impaction of the fluid dragging force on entrapped magnetic particles. The magnet assembly consisting of two identical magnet disks shown in Figure 3 has the flux density of 0.8 T on the magnet surface as determined by a gauss meter. With such a magnet assembly a stable packed bed in the capillary is limited to about 5 cm in length. For the longer packed beds, it is difficult to maintain the integrity of the packing structure due to particle “bleeding”. This phenomenon is believed to be related to the behavior of the magnetic particles under the influence of competing forces. The dragging force arising from electroosmotic flow is the most important competing force opposing to the magnetic force acting on the particle. For the magnetic particles to be trapped in a flow channel, a necessary condition imposed is to make the magnetic force (Fmag) dominate over the competing drag force (Fdrag). With the experimental setup employed in this work, this condition can be met for the initial short packing structure, but an upset is bound to occur with a further increase in the packed bed length. This is due to the fact that the total magnetic force (ΣFmag) acting on the particle assembly weakens markedly with increasing distances, whereas the total fluid drag force (ΣFdrag) is enhanced with increasing number of particles. At some critical point, the fluid drag force will outweigh the magnetic force, resulting in destabilization and degradation of the packed bed. Modifications to the experimental setup with respect to the configuration of applied magnetic fields are currently under investigation in this laboratory in attempt to overcome the limits imposed on column length. Reversed-Phase CEC Separations. For the reasons give above, we were unable to prepare full-length packed capillary columns using the experimental setup described in this work. Nevertheless, capillary columns with packed sections were used to separate neutral compounds with a view to test the feasibility of the magnetically immobilized beds for electrochromatography. Figure 4 shows the separations of two neutral compounds, thiourea and anthracene, achieved in columns packed with magnetic particles of diameters of 3.0 (Figure 4a) and 1.0 µm (Figure 4b), respectively. Although the packed beds of the two columns are of limited length (2.5 and 1.8 cm, respectively), the pair of neutral compounds are baseline separated within 15 min. It is noted that, with a capillary of total length being 50 cm, less than 5% of the column length is used for separation function. As such, there is ample room for reduction of the analysis time should a capillary column of shorter length be used. Unfortunately, the commercial CE instrument used in this work requires the total length of the capillary not less than 50 cm to facilitate its operating. Since thiourea is unretained under our experimental conditions, its retention time is taken as dead time for evaluation of retention behavior of a given chromatographic system. The retention time, tR, the retention factor, k, the plate number, N, the plate height, H, and the reduced plate height, h, of the analytes separated in packed columns are listed in Table 1. The column (21) Oleschnuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal. Chem. 2000, 72, 585-590.
Figure 4. Reversed-phase CEC separation of thiourea and anthracene. (a) Column, 250 µm i.d. × 50 cm (2.5 cm section packed with 3.0 µm ODS magnetic particles); mobile phase, 5 mM phosphate buffer (pH 8.0) containing 80% methanol; applied voltage, 15 kV; detection, 254 nm. (b) Column, 250 µm i.d. × 50 cm (1.8 cm section packed with 1.0 µm ODS magnetic particles); other parameters are the same as for (a). Table 1. Retention Characteristics and Column Efficiency for Two Magnetically Immobilized Bedsa column
analyte
tR (min)
k
N/m
H (µm)
h
1
thiourea anthracene
11.67 13.26
0.00 0.14
174 520 156 880
5.73 6.37
1.91 2.12
2
thiourea anthracene
8.14 9.00
0.00 0.09
273 889 359 389
3.65 2.78
3.65 2.78
a Chromatographic conditions are as listed in the caption of Figure 4.
packed with 3 µm particles exhibits greater retention times and fewer plate numbers compared with those of the column packed with 1 µm particles. This is to be expected from general chromatographic theory that analyte bands are more retarded and more dispersed in a longer bed packed with larger particles. Coming to the retention factors and reduced plate heights, it is however notable that there exist some discrepancies between theoretical expectations and experimental observations. First, the retention factor of anthracene is 50% higher with the column Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
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Figure 5. Separation of a mixture containing thiourea, toluene, naphthalene, fluorine, and anthracene (in the order of elution). Column, 250 µm i.d. × 50 cm (5 cm section packed with 3.0 µm ODS magnetic particles); mobile phase, 5 mM phosphate buffer (pH 8.0) containing 70% methanol; other parameters are the same as for Figure 4.
packed with 3 µm particles. Assuming the particles of different sizes are of the same surface area, they are expected to give rise to the same retention factor for a given analyte. This is because, by definition, the retention factor is proportional to the distribution coefficient of the analyte and the phase ratio of the packing material. The greater retention with 3 µm particles suggests a higher phase ratio arising from dense chemical bonding. Second, the reduced plate heights are 30-90% higher with the column packed with 1 µm particles, indicating that the packing structure and the flow profile are not as homogeneous as those with 3 µm (22) Gottschlich, N.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal. Chem. 2001, 73, 2669-2674. (23) Throckmorton, D. J.; Shepodd, T. J.; Singh, A. K. Anal. Chem. 2002, 74, 784-789. (24) Reichmuth, D. S.; Shepodd, T. J.; Kirby, B. J. Anal. Chem. 2005, 77, 29973000. (25) Ericson, C.; Holm, J.; Ericson, T.; Hjerten, S. Anal. Chem. 2000, 72, 8187.
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particles. Despite the fact that further studies are needed to clarify these issues, the results with these columns confirm that dense uniform packing structure comprised of magnetically responsive packing material is attainable with an external magnetic field applied orthogonally to the electroosmotic flow. Having established the feasibility of magnetically immobilized beds for electrochromatography, we proceeded to attempt more complex separations with a longer packed bed. Figure 5 shows CEC separation of five neutral compounds on a 5 cm bed packed with 3 µm particles. Thiourea was included as a neutral marker for electroosmotic velocity, and the other four compounds were selected as a representative sample containing aromatic hydrocarbons. As can be seen from the chromatogram, the five compounds are nearly baseline separated, with retention factor (given in parentheses) increasing in the order of thiourea (0), toluene (0.186), naphthalene (0.335), fluorine (0.895), and anthracene (1.432). Apparently, separation of aromatic hydrocarbons is entirely based on their differential interactions with the hydrophobic ligands bonded to magnetic silica particles. In conclusion, the preliminary results reported here demonstrate that magnetically responsive packing material can be entrapped in a flow channel using an external magnetic field. The particle assembly thus formed can be further impacted by electroosmotic flow into a uniform packing structure capable of delivering high column efficiency for chromatography. A distinct advantage of this approach over conventional methods is that the applied magnetic field provides an additional control over the localized placement of separation media in flow channels, thus opening up new possibilities for construction of advanced chipbased chromatographic systems with complex architectures comprising curved and intersecting channels.22-25 ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (20375027, 20575044).
Received for review February 11, 2007. Accepted April 25, 2007. AC070288B
