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Electrokinetic Effects in Poly(ethylene glycol)-Coated Capillaries Induced by Specific Adsorption of Cations Detlev Belder* and Jo¨rg Warnke Abteilung fu¨ r Chromatographie, Max-Planck-Institut fu¨ r Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mu¨ lheim an der Ruhr, Germany Received January 22, 2001. In Final Form: May 25, 2001 Capillaries coated with poly(ethylene glycol) (PEG) exhibit unique electrokinetic properties because of the specific adsorption of cations at the solid/liquid interface. The adsorption of cations present in methanolor acetonitrile-containing electrolytes can induce a positive surface charge on PEG-coated capillaries. This results in an adjustable anodic electroosmotic flow (EOF) in nonaqueous electrolytes, whereas a reduced cathodic EOF is observed in aqueous electrolytes. The EOF is dependent on the electrolyte constitution, namely, on the type of solvent used and especially on the abilities of the background cations to interact with polyethers such as PEG. The dependency of the EOF on the electrolyte concentration in nonaqueous electrolytes can be explained by two counterbalancing mechanisms: (i) the increase in surface charge density and (ii) the decrease in double-layer thickness. Using different alkali metal and alkaline earth metal cations dissolved in methanol the EOF can be varied over a wide range. The magnitude of the induced anodic EOF was found to vary in the order Ba2+ > Ca2+ > Mg2+ for alkaline earth metal ions and K+ > Cs+ > Na+ for alkali metal ions, whereas with lithium ions, a cathodic EOF was observed. The magnitude of the anodic EOF of PEG-coated capillaries in nonaqueous electrolytes was found to be dependent on the coating thickness.
1. Introduction Hydrophilic polymers such as poly(vinyl alcohols) (PVAs), polyethers, and cellulose derivatives are widely used as surface coating materials for different chemical, biotechnical, and biomedical applications. Important desired properties of surfaces coated with hydrophilic polymers are the reduction of protein and cell adsorption and improved wettability, which have led to the application of these materials as biocompatible surfaces, e.g., for catheters, contact lenses, etc.1 Especially coatings based on poly(ethyleneglycol) (PEG) as polyethers have found widespread applications in analytical chemistry, e.g., for the generation of polar stationary phases for gas chromatography, for biosensor applications, and for capillary electrophoresis (CE). In capillary electrophoresis, nonionic hydrophilic polymers are often used as coating materials for the capillary walls to reduce electroosmosis and adsorption of analytes.2-7 Control of electroosmosis is an important task in electrophoretic separation techniques, as the magnitude and direction of the EOF affect the net mobilities of analytes in the system. The electroosmotic flow (EOF) is related to the electric double layer at the solid/liquid interface and is dependent on the physicochemical properties of the surface and on the electrolyte composition. Water is used as the “normal” solvent constituting the electrolyte in CE. The electroosmotic flow characteristics * Author to whom correspondence should be addressed. Tel.: ++49 208 2275. Fax: ++49 208 2982. E-mail:
[email protected]. (1) Harris, J. M., Ed. Poly(ethylene glycol) Chemistry; Plenum Press: New York, 1992. (2) Bruin, G. J. M.; Chang, J. P.; Kuhlman, R. H.; Zegers, K.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1989, 471, 429. (3) Hjerten, S. J. Chromatogr. 1985, 347, 191. (4) Ng, C. L.; Lee, H. K.; Li, S. F. Y. J. Liq. Chromatogr. 1994, 17, 3847. (5) Gilges, M.; Klemiss, M. H.; Schomburg, G. Anal. Chem. 1994, 66, 2038. (6) Malik, A.; Zhao, Z.; Lee, M. L. J. Microcolumn Sep. 1993, 5, 116. (7) Belder, D.; Sto¨ckigt, D. J. Chromatogr. A 1996, 752, 271.
of the usually employed uncoated fused-silica (FS) capillaries in these aqueous systems are well-known. The EOF of fused-silica capillaries is directed to the cathode, and the magnitude is dependent on the buffer pH and concentration.8-10 The electroosmotic flow can be manipulated by the addition of surface-active compounds to the electrolyte and also by the application of radial electrical fields.11-13 A very efficient way to modify the EOF, often applied in aqueous CE, is chemical modification of the capillary surface by the application of appropriate surface coatings. The electroosmotic flow can be increased, reduced, or even reversed using differently charged coatings. With nonionic hydrophilic coated capillaries, such as those coated with PVA and PEG,14-16 a reduced electroosmotic flow is observed that is influenced by the aforementioned parameters to a much lower extent. Hence, complex rinsing steps and preconditioning steps in stabilize the EOF are usually not required as they would be for bare FS capillaries. Although nonaqueous media have been applied in capillary electrophoresis,17-25 CE separations in non(8) Lambert, W. J.; Middleton, D. L. Anal. Chem. 1990, 62, 1585. (9) Lukacs, K. D.; Jorgenson, J. W. J. High Resolut. Chromatogr. 1985, 8, 407. (10) McCormick, R. M. Anal. Chem. 1988, 60, 2322. (11) Lee, C. S.; Blanchard, W. C.; Wu, C. T. Anal. Chem. 1990, 62, 1550. (12) Hayes, M. A.; Ewing, A. G. Anal. Chem. 1992, 64, 512-516. (13) Belder, D.; Schomburg, G. J. High Resolut. Chromatogr. 1992, 15, 686. (14) Iki, N.; Yeung, E. S. J. Chromatogr. A 1996, 731, 273. (15) Huang, M.; Vorkink, W. P.; Lee, M. L. J. Microcolumn Sep. 1992, 4, 135. (16) Burns, L. N.; Van Alstine, J. M.; Harris, J. M. Langmuir 1995, 11, 2768. (17) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135. (18) Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141. (19) Chiari, M.; Kenndler, E. J. Chromatogr. A 1995, 716, 303. (20) Jansson, M.; Roeraade, J. Chromatographia 1995, 40, 163. (21) Okada, T. J. Chromatogr. A 1995, 695, 309. (22) Bjornsdottir, I.; Hansen, S. H. J. Chromatogr. A 1995, 711, 313. (23) Benson, L. M.; Tomlinson, A. J.; Reid, J. M.; Walker D. L.; Ames, M. M.; Naylor, S. J. High Resolut. Chromatogr. 1993, 16, 324.
10.1021/la010115w CCC: $20.00 © 2001 American Chemical Society Published on Web 07/12/2001
Electrokinetic Effects in PEG-Coated Capillaries
aqueous electrolytes have mainly been performed with uncoated capillaries. In such systems, the electroosmotic flow has been studied26-30 and found to be rather unstable, as in the case of aqueous electrolytes. Recently, we extended the applicability of coated capillaries to a directed control of electroosmosis in nonaqueous CE.31,32 The electrokinetic properties of the employed hydrophilic nonionic capillary coatings based on either poly(ethylene glycol) or poly(vinyl alcohol) on the EOF was quite different. For PVA-coated capillaries, a reduced cathodic EOF was observed as expected, whereas a reversed EOF was observed with PEG-coated capillaries if methanol- or acetonitrile-based electrolytes were used. The reversal of the direction of the EOF is related to an induced positive charge on the capillary surface and was explained by the complexation of cations by the poly(ethylene glycol) coating, which can formally be described by the equation
The interaction of poly(ethylene glycol)s as polyethers with cationic species is closely related to cation complexation by crown ethers.33-35 In a recent work, we demonstrated that the EOF in nonaqueous CE can be controlled in a directed manner by the use of different alkaline earth metals as background electrolytes.36 In the present contribution, we studied the influence of the concentration of different alkali and alkaline earth metal ions on the electrokinetic properties of PEG coatings. Furthermore, we studied the influence of the coating thickness of PEG on the cation-induced electroosmotic flow and the electrokinetic properties of capillary surfaces coated with nonbonded PEG37 in methanolic electrolytes. 2. Experimental Section 2.1. Reagents. All samples, solutions, and buffers were prepared using analytical-grade chemicals. HPLC-grade methanol; calcium chloride; barium chloride; and potassium, sodium, (24) Tjornelund, J.; Bazzanella, A.; Lochmann, H.; Ba¨chmann, K. J. Chromatogr. 1998, 811, 211. (25) Riekkola, M.-L.; Jussila, M.; Porras, S. P.; Valko, I. E. J. Chromatogr. A 2000, 892, 155. (26) Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801. (27) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59, 487. (28) Stevens, T. S.; Cortes, H. J. Anal. Chem. 1983, 55, 1365. (29) Wright, P. B.; Lister, A. S.; Dorsey, J. G. Anal. Chem. 1997, 69, 3251. (30) Valko, I. E., Siren, H.; Riekkola, M. L. J. Microcolumn Sep. 1999, 11, 199. (31) Belder, D.; Husmann, H.; Elke, K. J. Microcolumn Sep. 1999, 11, 209. (32) Belder, D.; Elke, K.; Husmann, H. J. Chromatogr. A 2000, 868, 63. (33) Banka, P. A.; Selser, J. C.; Wang, B.; Shenoy, D. K.; Martin, R. Macromolecules 1996, 29, 3956-3959. (34) Quina, F.; Sepulveda, L.; Sartori, R.; Abuin, E. B.; Pino, C. G.; Lissi, E. A. Macromolecules 1986, 19, 990. (35) Okada, T. J. Chromatogr. A 1999, 834, 73.
Langmuir, Vol. 17, No. 16, 2001 4963 and magnesium acetate were purchased from Merck (Darmstadt, Germany). Ammonium, lithium, and cesium acetate were purchased from Fluka (Buchs, Switzerland). The water content of methanol was about 0.3% (determined by GC). 2.2. Capillaries and Coatings. Fused-silica capillaries of 50 µm inner diameter were obtained from Polymicro Technologies (Phoenix, AZ). The PEG coating was applied by a static coating method38,39 similar to that used for the preparation of capillaries for gas chromatography by using the PE-1M-100 coating material [MW 1000, branched poly(ethylene glycol), liquid at 25 °C] from Innophase Corp. (Westbrook, CT). For that purpose, 0.5, 1, or 2% (w/w) of the coating material was dissolved in a solvent mixture of 50% (v/v) pentane and 50% (v/v) dichloromethane. This coating solution was introduced into a capillary that was about 4 m in length. After one end of the capillary had been sealed, the capillary was placed in a thermostated water bath at 25 °C, while the open end of the capillary was connected to an evacuated (0.5 bar) flask, effecting a slow evaporation of the solvent in the capillary. After complete evaporation of the solvent, the residual coating was immobilized. The coating material from Innophase contained an unknown cross-linking agent that is activated by heating as in the case of the thermal peroxide treatment described in the literature.40 For immobilization of the coating, the column was placed in a GC oven and heated to 220 °C, while the capillary was flushed with nitrogen at a pressure of 1.5 bar. Afterward, the capillary was rinsed with dichloromethane to remove residual non-cross-linked coating material, followed by another heating step for 4 h in a GC oven at 180 °C. The homogeneity and quality of the coating were then assessed by two independent methods. First, the capillaries were used as GC columns performing separations of a GC test mixture (Grob test) to reveal that the columns were uniformly coated. In the case of partial coatings, decreased capacity factors and/or separation efficencies for the compounds in the test mixture would be observed. Afterward, the capillaries were tested for their use in CE, by performing separations of basic proteins that have been described previously.28 2.3. EOF Measurements. Experiments were performed using an automated CE instrument. A freshly installed capillary was first flushed with water for 5 min and then rinsed with methanol for 5 min. Between a series of electrophoretic EOF determinations, the capillary was rinsed for 30 s with the respective electrolyte. The electroosmotic flow was determined from the migration time of a neutral UV-active compound. For this purpose, about 1 µL of DMSO was added to the outlet electrolyte (2 mL), and a voltage of (30 kV was applied until the migrating front reached the detector from the short end of the capillary. The total length of the capillaries used for EOF determinations was 48.5 cm, while the length to the detection window was 40 cm. UV detection was performed at 210 nm. 2.4. Instrumentation. CE experiments were performed with laboratory-made equipment with UV detection that has been described previously13 and with commercial automated HP Instruments (Waldbronn, Germany).
3. Results and Discussion PEG-coated capillaries exhibit unusual electrokinetic properties. Whereas only a very weak cathodic EOF is observed in typical aqueous buffers, the electroosmotic flow is reversed in several nonaqueous electrolytes. This behavior can be attributed to the selective adsorption of cations at the PEG/electrolyte interface.29,33 The dependency of the EOF on the concentration or ionic strength is quite different if the capillaries are operated with aqueous or nonaqueous electrolytes. For a discussion of the influence of ionic strength on the EOF, three equations will be introduced. (36) Belder, D.; Husmann, H.; Warnke, J. Electrophoresis 2000, 22, 666. (37) Preisler, J.; Yeung, E. S. Anal. Chem. 1996, 68, 2885. (38) Grob, K.; Grob, G. J. Chromatogr. 1981, 213, 211. (39) Sandra, P.; Redant, G.; Schacht, E.; Verzele, M. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1981, 8, 411. (40) Sandra, P.; Van Roelenbosch, M.; Temmerman, I.; Verzele, M. Chromatographia 1982, 16, 63.
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Figure 1. EOF of an uncoated and a PEG-coated capillary affected by the ammonium concentration of aqueous electrolytes. Each data point reflects the averaged data from five EOF determinations
According to the Smoluchowski equation
µEOF ) -
0ζ η
(2)
the electroosmotic mobility (µEOF) is dependent on the ζ potential (ζ), the dielectric constant (), and the viscosity (η). The ζ potential, the potential at the shear plane, is dependent on the double-layer thickness κ-1 and the surface charge density σ, as shown in eq 3.
σ 0κ
ζ)
(3)
The inverse double-layer thickness κ, which is also called Debye-Hu¨ckel parameter, is related to the ionic strength I according to the equation
κ)
x
2F2 I 0RT
(4)
where F is the Faraday constant and R is the universal gas constant. According to eqs 2-4, the electroosmotic mobility µEOF should be inversely proportional to the square root of the ionic strength I. Although this relationship is not exact in the case of the presence of a wall adsorption equilibrium of buffer cations,41,42 it accounts for the usual observed decrease in electroosmotic mobility with increasing ionic strength in uncoated bare FS capillaries. This “normal” dependency of the EOF on buffer concentration is also found for PEG-coated capillaries operated in aqueous ammonium acetate electrolytes. In Figure 1, plots of EOF vs ammonium acetate concentration are shown for an uncoated capillary compared to a PEG-coated (41) Salomon, K.; Burgi, D. S.; Helmer, J. C. J. Chromatogr. A 1991, 559, 69. (42) Thormann, W.; Zhang, C. X.; Caslavska, J.; Gebauer, P.; Mosher, R. A. Anal. Chem. 1998, 70, 549.
Figure 2. EOF of PEG-coated capillaries versus ammonium acetate concentration using different solvents. Each data point reflects the averaged data from five EOF determinations.
capillary. It is notable that not only the EOF of the PEG capillary is significantly reduced but also the EOF is more stable in the case of the coated capillary, as indicated by the error bars (n ) 5). When PEG-coated capillaries are operated with a methanolic ammonium acetate electrolyte instead, the direction of the EOF is reversed, and the electroosmotic mobility increases with increasing ammonium concentration; this is shown in Figure 2. The increase in magnitude of the EOF with ammonium concentration is rather significant at low concentrations, whereas at concentrations higher than 20 mmol L-1, only a weak increase in the EOF is observed. In the case of acetonitrile-based electrolytes, the increase in electroosmotic mobility with ammonium concentration is even more pronounced, and a maximum EOF value is observed at a concentration of about 8 mmol L-1. The slopes of the electrokinetic curves obtained with methanol- and acetonitrile-based electrolytes can be interpreted in terms of two concurrent mechanisms responsible for the generation of electroosmosis. With increasing ammonium concentration or ionic strength, the double-layer thickness decreases according to eq 4, which should result in a decrease in the EOF according to eqs 3 and 2. This effect is counterbalanced as the surface charge density σ, which is not constant as is often assumed, increases with increasing ammonium concentration. The increase in the surface charge density is caused by the adsorption of ammonium at the PEG surface. The impact of the ammonium concentration on the EOF is more pronounced in acetonitrile-based electrolytes compared to methanol-based electrolytes because the ions are less dissociated and stabilized in the solution and exhibit a higher affinity to the PEG surface. At higher concentrations when saturation of the PEG surface with ammonium ions occurs, the decreasing double-layer thickness, κ-1, causes a decrease in electroosmotic mobility. The electrokinetic curves differ significantly when various cations with different abilities to interact with polyethers such as PEG are used as background electrolytes. We prepared methanolic solutions with ammonium and different alkali and alkaline earth metal ions as background electrolytes and determined the dependency of the EOF on the electrolyte concentration in PEG-coated
Electrokinetic Effects in PEG-Coated Capillaries
Figure 3. Concentration of different alkali (dotted line) and alkaline earth metal ions (solid line) dissolved in methanol versus the EOF observed using PEG-coated capillaries.
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Figure 4. Comparison of the influence of the ammonium concentration of methanolic electrolytes on the EOF of permanently PEG-coated capillaries of different coating thicknesses and of a dynamically coated capillary. Each data point reflects the averaged data from five EOF determinations
capillaries. In Figure 3, the EOF values obtained for the different cations constituting the electrolytes are shown. For all alkali metal ions other than lithium, for all alkaline earth metal ions, and for ammonium, an anodic EOF is obtained. The different electrokinetic curves can be explained by the different abilities of the cations to interact with polyethers such as PEG. The complexation constants for PEG oligomers (n ) 13) with alkali metal ions in methanol found in the literature43 are in the order K+ > Cs+ . NH4+ > Na+ and correlate with the EOF values determined in our experiments. The larger the complexation constants, the larger the negative EOF values induced. According to the electrokinetic data shown in Figure 3, the complexation constants for the alkaline earth metals should vary in the order Ba2+ > Ca2+ > Mg2+. Complexation constants for the alkaline earth metal ions with polyethers such as PEG in methanolic solutions were not found in the literature. However, in a recent competitive lithium-7 NMR study on the formation constants of complexes of metal ions with the macrocyclic polyether 18-crown-6 in acetonitrile, a similar order was found.44 The different slopes of the electrokinetic curves for the alkali and the alkaline earth metal ions in Figure 3 can
be explained by a reduction in electroosmotic mobility with increasing ionic strength. The decrease in the doublelayer thickness with increasing concentration is much more pronounced in a 1:2 electrolyte (MX2) than a 1:1 electrolyte (MX); therefore, the EOF increases more strongly with ionic strength for the alkaline earth metal ions. As the interaction of cations with poly(ethylene glycol) immobilized on capillary surfaces could be revealed by the induced electrokinetic effects, we wondered whether the thickness of the PEG coating affects the adsorption and by that electroosmosis. To investigate this issue, we prepared capillaries with different coating thicknesses. As the PEG coating is generated by a static coating method, this can easily be accomplished by using coating solutions with different contents of PEG. The generated coating thickness can be determined in GC experiments through the phase ratio, an approach that is commonly used for the characterization of stationary phases in gas chromatography.45 Whereas we used coating solutions of 0.5% PEG in a 1:1 mixture of pentane and dichloromethane for the generations of coatings for previous experiments, we employed coating solutions of 1 and 2% (w/w) in order to generate coatings of increased thickness. The film thick-
(43) Okada, T. J. Chromatogr. A 1999, 834, 73. (44) Shamsipur, M.; Madrakian, T. Polyhedron 2000, 19, 1681.
(45) Schomburg, G. Gas Chromatography: A Practical Course; VCH: Weinheim, Germany, 1990.
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ness were determined in GC experiments to be 0.03 µm with a 0.5% coating solution, 0.05 µm with a 1% coating solution, and 0.09 µm with a 2% coating solution. As shown in Figure 4, where the EOF is plotted vs the NH4OAc concentration, the induced anodic electroosmotic flow is dependent on the coating film thickness. At low electrolyte concentration, the electroosmotic mobility increases with film thickness, whereas the electroosmotic mobility remains at about the same value at higher buffer concentrations. The dependency of the EOF on the coating film thickness indicates that the ions are able to penetrate the coating and that the uptake of cations is dependent on the phase ratio. It should therefore be possible to use PEGbased stationary phases in electrochromatography using methanolic electrolytes for the separation of small cations. However, in some initial experiments, where we tried to use PEG-coated capillaries (i.d. ) 50 µm) for the separation of a mixture of metal ions in a methanolic electrolyte, the cations were not significantly retarded. The expected chromatographic effects would, of course, be much more pronounced if thinner capillaries or packed columns with much higher phase ratios were used. In Figure 4, we also plot for comparison the corresponding electrokinetic curve for a capillary surface that was dynamically coated with PEG, which can be described as a very thin nonbonded coating. For this purpose, 0.1%
Belder and Warnke
PEG (Carbowax, PEG 1000) was dissolved in the electrolyte. In the dynamically coated capillary, the EOF was directed toward the cathode at low ammonium concentration and toward the anode at concentrations higher than 20 mmol L-1. This shows that the affinity of PEG for the fused-silica surface is relatively low in methanolic solutions, and therefore, a cathodic EOF is observed in an uncoated capillary. With increasing ammonium concentration, positively charged PEG-ammonium associates are formed in solution, which are easily adsorbed onto the previously negatively charged FS capillary surface, resulting in a reversed anodic EOF at high ammonium concentrations. Whereas the dynamic modification of the capillary surface with PEG appears to be dependent on the electrolyte concentration, this was not observed in aqueous systems, where a reduced cathodic EOF was always found. In summary, we were able to show that the electroosmotic flow in poly(ethylene glycol)-coated capillaries operated in nonaqueous electrolytes is affected by the adsorption cations. Capillary electrophoresis is a powerful tool for studying the elektrokinetic properties of coatings especially the interactions of cations with immobilized and dissolved polyethers. LA010115W
