Capillaries Modified by Polyelectrolyte Multilayers for Electrophoretic

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Anal. Chem. 1999, 71, 4007-4013

Capillaries Modified by Polyelectrolyte Multilayers for Electrophoretic Separations Timothy W. Graul† and Joseph B. Schlenoff*

Department of Chemistry and Center for Materials Research and Technology (MARTECH), The Florida State University, Tallahassee Florida 32306-4390

Fused silica capillaries coated with thin films of physically adsorbed charged polymers are employed for capillary zone electrophoretic separations. The coating is a polyelectrolyte multilayer, constructed in situ by alternating rinses with positively and negatively charged polymers. The thickness of the multilayer and amount of surface charge is controlled by the concentration of salt in the deposition solutions. The direction of the electroosmotic flow oscillates as the multilayer surface charge alternates in polarity during buildup. The apparent surface charge, deduced from the electroosmotic mobility, is considerably less than the nominal surface charge of the film. Mechanisms limiting this apparent surface charge for a bufferpermeable layer are discussed. The multilayer-coated columns exhibit many desirable features in addition to ease of construction and reproducible control of electroosmotic flow: stable flow rates are achieved immediately on exposure of the column to running buffer, and reversed flow is possible. Columns are also found to be stable to extremes of pH and ionic strength, and to dehydration/rehydration. A series of basic proteins are separated with good efficiency, demonstrating column resistance to irreversible protein adsorption. Partitioning and separation of neutral solutes using thicker films is demonstrated. Control of charge and composition of the capillary wall is critical in regulating electroosmotic flow, EOF, in capillary electrophoretic separations.1,2 At the same time, the charge on the wall provides a mechanism for interaction and, potentially, adsorption of charged macromolecules such as proteins. Thus, although excellent separation is predicted for proteins in optimal cases,3 adsorption via electrostatic interactions, hydrogen bonding, hydrophobic patches, and biospecific sites compromises resolution.4 Of these mechanisms, Coulombic interactions and hydrogen bonding are often cited as the main contributors to adsorption.4-6 † Current address: Central Research Division, Pfizer Inc., Groton CT. (1) Weinberger, R. Practical Capillary Electrophoresis; Academic Press: London, 1993. (2) Jorgenson, J.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298. (3) Bushey, M. M.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 301. (4) El Rassi, Z.; Nashabeh, W. In Capillary Zone Electrophoresis of Biopolymers with Hydrophilic Fused-Silica Capillaries in Capillary Electrophoresis Technology; Guzman, N. A., Ed.; Marcel Dekker: New York, 1993; pp 383-434. (5) Liu, Q.; Lin, F.; Hartwick, R. A. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 707. (6) Towns, J. K.; Regnier, F. E. Anal. Chem. 1992, 64, 2473.

10.1021/ac990277l CCC: $18.00 Published on Web 08/17/1999

© 1999 American Chemical Society

Bushey and Jorgenson reported that capacity factors, attributable to electrostatic interaction, as low as 0.05 can reduce efficiencies 20-fold.3 Additionally, since adsorption modifies wall charge, the reproducibility of retention time can be severely impacted.7 Strategies employed to modify capillary walls, and thereby EOF and/or adsorption phenomena, have been several, although each can have disadvantages. Extremes of pH yielding highly negatively charged proteins (high pH, considerably beyond the protein isoelectric point)8,9 or weakly charged capillary walls (low pH)10,11 have been used to minimize wall/protein attractive interactions. Unfortunately, proteins tend to degrade at extremes of pH. Salt ions, when added in high concentration, compete effectively for adsorption sites,12 but joule heating is more serious.13 Dynamic modification by neutral or charge species has been reviewed recently by Corradini.14 Here, surface-modifying agents are employed in the background electrolyte. Again, there is the potential for protein denaturation. Dynamic modifiers particularly relevant to this work are polyamines and polyammonium salts.15-17 On adsorption, these polyelectrolytes induce capillary wall charge reversal which leads to reversed EOF. Irreversible surface modification with neutral18 or charged5,19-21 coatings offers an alternative to dynamic modification. A drawback is that multiple time-consuming steps are often required to form chemical bonds, and reactions at surfaces tend not to be as complete as they are in solution. The physical adsorption of polyelectrolytes has been offered as a rapid method of producing quasi-stable coatings.21-24 Although each repeat unit/silanol electrostatic bond is rather weak, a multiplicity of bonds ensures (7) Iki, N.; Yeung, E. S. J. Chromatogr. A 1996, 731, 273. (8) Lauer, H. H.; McManigill, D. Anal. Chem. 1986, 58, 166. (9) Lee, K. J.; Heo, G. S. J. Chromatogr. 1991, 559, 317. (10) McCormick, R. M. Anal. Chem. 1988, 60, 2322. (11) Vinther, A.; Bjorn, S. E.; Sorenson, H. H.; Soeberg, H. J. Chromatogr. 1990, 516, 175. (12) Lauer, H. H.; McManigill, D. Trend. Anal. Chem. 1986, 5, 11. (13) Green, J. S.; Jorgenson, J. W. J. Chromatogr. 1989, 478, 63. (14) Corradini, D.; J. Chromatogr. B 1997, 699, 221. (15) Cohen, N.; Grushka, E. J. Capillary Electrophor. 1994, 2, 112. (16) Madabhushi, R. S. Electrophoresis 1998, 19, 224. (17) Krokhin, O. V.; Hoshino, H.; Shpigun, O. A.; Yotsuyanagi, T. J. Chromatogr. A 1997, 776, 329. (18) Cobb, K. A.; Dolnik, V.; Novotny, M. Anal. Chem. 1990, 62, 2478. (19) Smith, J. T.; El Rassi, Z. Electrophoresis 1993, 14, 396. (20) Thorsteinsdottir, M.; Isaksson, R.; Westerlund, D. Electrophoresis 1995, 16, 557 (21) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 36, 126. (22) Wiktorowicz, J. E.; Colburn, J. C. Electrophoresis 1990, 11, 769. (23) Towns, J. K.; Regnier, F. E. J. Chromatogr. 1990, 516, 69. (24) Stathakis, C.; Cassidy, R. M. Anal. Chem. 1994, 66, 2110.

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stability of the coating. Cationic polyelectrolytes employed for this purpose include polyarginine,25 chitosan,26 poly(diallyldimethylammonium chloride),21 and polyethyleneimine.23 However, it has been documented that the electroosmotic mobility deteriorates during use, purportedly from “bleeding” of the coating.21,25,27 We have explored the mechanism of formation and charge balance in polyelectrolyte multilayers (PEMs)28-31 fabricated by a recently discovered layer-by-layer deposition process.32,33 In this technique, a substrate is exposed in an alternating fashion to solutions of oppositely charged macromolecules. The increment in film thickness per deposition step rapidly reaches a steady state value, permitting fine control over layer thickness. An essential feature of film growth is that the existing surface charge is actually overcompensated upon adsorption of oppositely charged polyelectrolyte.28,33 Thus, the sign of surface charge is reversed following each exposure to polymer, priming the layer for the next deposition step. Surface charge, and therefore layer thickness, is determined principally by the concentration of salt ions codissolved in the polyelectrolyte solutions. The magnitude of the surface excess charge can be measured directly with radioanalytical methods.28,34,35 In this paper we describe the application of these novel ultrathin films to analytical separations. There are many attractive features to the use of PEMs: their preparation is straightforward and reproducible, and one can mix synthetic and natural (e.g., proteins)36 polyelectrolytes. As we will demonstrate, the result is a very efficient and versatile system for regulating wall capillary wall charge and affinity for proteins. The multilayer system described here is similar to a 3-layer polyelectrolyte coating recently used for capillary electrophoresis separations.37 EXPERIMENTAL SECTION Poly(diallyldimethylammonium chloride), PDADMAC (Aldrich, Mw ) 250 000-400 000, Mw/Mn ) 2.9) and poly(styrene sulfonate), sodium salt, PSS (Scientific Polymer Products, Mw ) 6 × 106) were purified by extensive dialysis against distilled water using 12 000-14 000 molecular-weight-cutoff dialysis tubing (Allied-Fisher Scientific). Basic proteins R-chymotrypsinogen A (type II from bovine pancreas), ribonuclease A (type XII-A from bovine pancreas), cytochrome c (from bovine heart), and lysozyme (grade I from chicken egg white) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Protein sample concentrations before injection were either 0.25 or 0.55 mg mL-1. All other chemicals were used as received from Allied-Fisher. Separations were performed on a Beckman P/ACE System 2100 capillary electrophoresis unit (Palo Alto, CA) with UV (25) Chiu, R. W.; Jimenez, J. C.; Monnig, C. A. Anal. Chim. Acta 1995, 307, 193. (26) Sun, P.; Landman, A.; Hartwick, R. A. J. Microcolumn Sep. 1994, 6, 403. (27) Cunico, R. L.; Gruhn, V.; Kresin, L.; Nitecki, D. E.; Wiktorowicz, J. E. J. Chromatogr. 1991, 559, 567. (28) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626. (29) Laurent, D.; Schlenoff, J. B. Langmuir 1997, 13, 1552. (30) Schlenoff, J, B.; Li, M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 943. (31) Dubas, S. T.; Schlenoff, J. B. Macromolecules, submitted for publication. (32) Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, 160. (33) Decher, G. Science 1997, 277, 1232. (34) Li, M.; Schlenoff, J. B. Anal. Chem. 1994, 66, 824. (35) Graul, T. W.; Li, M.; Schlenoff, J. B. J. Phys. Chem. 1999, 103, 2718. (36) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (37) Katayama, H.; Ishihama, Y.; Asakawa, N. Anal. Chem. 1998, 70, 5272.

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detection. Fused silica capillary with 50 µm i.d., 360 µm o.d., and polyimide outer coating was purchased from Polymicro Technologies (Phoenix, AZ). Multilayer coatings were deposited using the rinse function (rate of 250 cm min-1 linear velocity, 5 µL min-1 volume flow rate) on the Beckman CE system. Polymer deposition solutions contained 10 mM polymer, and varying NaCl concentration (polymer concentrations are based on the repeat unit). The capillary was first conditioned by a 30-min rinse of 1 M NaOH. Then water was flushed through the capillary for 3 min. The first monolayer of polymer (PDADMAC) was deposited by rinsing the polymer solution through the capillary for 20 min followed by a 5-min water rinse. All other polymer depositions were done with 5-min rinses followed by 5-min water rinses. The multilayer coatings used for the protein separations and reproducibility studies consisted of 6.5 layer pairs (a layer pair is a layer of cationic polymer plus a layer of anionic polymer, also termed a “bilayer” in other studies on PEMs) where the first 3.5-layer pairs were deposited with no salt and the last 3 with 0.5 M NaCl. The running electrolyte for electrophoresis experiments was pH 4.0-8.0 phosphate buffer at various concentrations. Electrolyte solutions were made by adding 20 mM solutions of phosphoric acid to 20 mM phosphate salt solutions until the proper pH was achieved. The capillary length was 37 cm, length to detector was 30 cm, and the applied voltage was typically 15 kV. UV detection was done at 254 or 214 nm and injection was performed electrokinetically (5 kV for 5 s, ∼5 nL volume). Acetone was used as a neutral electroosmotic flow marker, and 2-phenoxypropionic acid (2-PPA) and lidocaine were used as negative and positive markers, respectively. Standard deviation values are reported as (1σ. Electroosmotic mobility (µeo) is used here to quantify the electroosmotic flow (EOF) and is given as the velocity of solvent flow per unit electric field strength (cm2 V-1 s-1). RESULTS AND DISCUSSION Single Layer of Adsorbed Polyelectrolyte. The adsorption of a single layer of PDADMAC induces reversed EOF. We performed an extensive study on the dependence of separation behavior on polymer deposition conditions. Our findings, which will be presented in detail elsewhere,38 are generally similar to those previously reported21,26 for adsorbed cationic polyelectrolytes for capillary zone electrophoresis, CZE. The separation of some representative basic proteins is depicted in Figure 1. Separations were performed at pH 4.0 and 6.0 (near-physiological). The relevant theoretical plate numbers for the two pH values are given in Table 1. Although baseline separation was achieved with all proteins, significant tailing was observed. In addition, µeo drifted toward lower values with time, especially at the higher pH. Using radiolabeled polymers, this drift was found to be a result of slow conformational changes of polymer, and not to bleeding (spontaneous desorption) of the polymer from the column.38 Other studies with radiolabeled polyelectrolytes in PEMs similar to those used here have shown no evidence for spontaneous desorption in the presence or absence of salt.28,30 Exchange of surface-bound polymers for solution polymers occurs to the extent of a few percent but only after several days.28,31 As with earlier studies, it (38) Graul, T. W.; Schlenoff, J. B., manuscript in preparation.

Figure 1. Separation at pH 4.0 of four basic proteins: (1) R-chymotrypsinogen A, (2) ribonuclease A, (3) cytochrome c, and (4) lysozyme. Single PDADMAC layer deposited on capillary from 10 mM polymer solution in 3 × 10-3 M NaOH. Prime number labels indicate impurities or degradation products. Conditions: 20 mM phosphate running buffer, 2-phenoxypropionic acid used as negative marker (first peak), capillary length 37 cm (30 cm to detector), 50-µm i.d. column, 12-kV applied voltage, electrokinetic injection (5 kV for 5 s), 214 nm detection. Table 1. Migration Times and Theoretical Plate Numbers from Separation of Four Basic Proteins with Layer of Physically Adsorbed PDADMAC

protein R-chymotrypsinogen A ribonuclease A cytochrome c lysozyme

migration theoretical migration theoretical times (min) plates (N) times (min) plates (N) at pH 4.0 at pH 4.0 at pH 6.0 at pH 6.0 5.60 ( .01 5.92 ( .01 7.42 ( .02 8.77 ( .02

62 800 76 200 49 200 25 500

5.89 ( .01 6.14 ( .01 7.72 ( .02 9.36 ( .02

56 200 65 900 45 100 29 200

was found that a rinse with fresh polyelectrolyte solution prior to each run restored the flow rate. Multilayer Deposition: Electroosmotic Flow and Implications for Surface Charge. Multilayers were prepared using commercially available strongly charged polyelectrolytes: PSS and PDADMAC. The driving forces for the formation of multilayers using these two polymers were explored in a recent study using robotic deposition onto spinning silicon wafers.31 Variables such as deposition time, polymer concentration, salt concentration, salt type, and solvent quality were assessed. The critical role of salt in controlling polymer adsorption via an ion-exchange competition was proposed. In general, multilayers may be deposited on a variety of hydrophilic substrates33,39 and other variables, such as polymer concentration, deposition time, and molecular weight, have little impact on film thickness. In our experiments,31 where the substrate was the native oxide layer on a silicon wafer, the (39) Decher, G. In Comprehensive Supramolecular Chemistry; Sauvage, J. P., Hosseini, M. W., Eds.; Pergamon Press: Oxford, 1996.

Figure 2. Capillary electrophoretic examination of a multilayer buildup from 0.5 M NaCl solutions. PDADMAC and PSS were alternately deposited. Sodium hydroxide (3 × 10-3 M) was included in the first PDADMAC deposition solution only. Acetone was used as a neutral marker, with detection at 254 nm. Fifteen kV applied voltage. All other separation conditions were identical to Figure 1.

dependence of the layer pair thickness (tlp, Å) on salt concentration, M (mol L-1), was approximately linear and followed the trend40

tlp ) 25 + 165M

(1)

By assuming oxidized silicon represents an appropriate surrogate surface for fused quartz, the thickness of the PEM coating on the capillary walls is thus controlled with high precision ((2%) by salt. Capillary electrophoresis with no salt added to polyelectrolytes during multilayer buildup showed flow reversal with positive outer layers, but the electroosmotic mobility was not reproducible. Much greater reproducibility in µeo was obtained from multilayers made in the presence of salt. Figure 2 demonstrates the magnitude of the electroosmotic mobility and its dependence on the number of polymer layers deposited on the capillary wall. Several points are noteworthy: first, as expected, the direction of EOF depends on the sign of surface charge. We use the convention that the “normal” direction, from anode to cathode, is positive. The EOF oscillates in an extremely uniform manner, depending on the sign of the surface charge, after the first layer. This is reminiscent of recent ζ-potential measurements on PEM-coated particulate suspensions, as determined by electrophoresis.41 A clear difference of polymer-coated vs bare silica (zero-layer pairs) is that the EOF is enhanced in the former over the latter at this pH. The RSD of the µeo values between the first six bilayers (Figure 2) is 0.5%. It is not surprising to find a higher µeo for multilayer-coated capillary compared to bare silica, where, at pH 4, the silanol groups are minimally ionized. Faster EOF from higher wall charge is (40) It should be noted that the thickness is highly dependent on the choice of polymers. For example, Lo¨sche et al. (Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwman W. G.; Kjaer, K. Macromolecules 1998, 31, 8893) determined, for PSS and poly(allylamine hydrochloride), tlp (Å) ) 28 + 16M. (41) Caruso, F.; Donath, E.; Mohwald, H. J. Phys. Chem. B 1998, 102, 2011

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Figure 3. Capillary electrophoretic examination of a PDADMAC/ PSS multilayer buildup deposited with 0.1 M NaCl solutions for the first layer pair, 0.2 M NaCl for the second, 0.5 M NaCl for the third, and 1.0 M NaCl for the fourth layer pair. Separation conditions as in Figure 2.

expected following basic descriptions of electrophoretic flow:

µeo )

σκ-1 η

(2)

where µeo is the electrophoretic mobility (m2 V-1 s-1), σ (C m-2) is the surface charge density, and η the viscosity of solution (N s m-2). κ (m), the inverse of the Debye-Hu¨ckel parameter, is the Debye length (roughly, the double layer thickness), which depends on ionic strength, I, according to the following:

κ)F

[

2I 0rRT

]

1/2

(3)

F is the Faraday constant (96 495 C mol-1), R the gas constant (8.31 J K-1 mol-1), r the dielectric constant of the buffer (78 for water), and 0 the permittivity of a vacuum (8.85 × 10-12 C2 N-1 m-2). Electroosmotic Mobility is Independent of Polymer Surface Charge. From eq 2, one would expect that PEMs with thicker layers, thus high excess surface charge, should yield faster µeo. It is possible to recast eq 1 in terms of charge density, or, more precisely, projected areal charge density, σsurf, of excess polymer charge (also termed “chemical” charge density). Using 310 g mol-1 as the molecular weight of a PDADMAC/PSS ion pair, assuming the density is 1.1 g cm-3, the degree of charge overcompensation is 50% of the charge of a layer pair,28 and the layers contain 15 wt % water42

σsurf (C m-2) ) 0.36 + 2.4M

(4)

In reality, µeo is independent of σsurf over a wide range of M. This (42) Farhat, T.; Yassin, G.; Dubas, S. T.; Schlenoff, J. B., Langmuir submitted for publication.

4010 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

is illustrated in Figure 3, which depicts electrophoresis measurements from a multilayer where each subsequent layer is deposited from higher salt concentration. Since a dry monolayer of PDADMAC/PSS ion pairs represents about 0.27 C m-2, clearly the concept of a “surface charge” in PEMs is more complex. The excess charge is actually distributed over several layers of polymer. Before a more detailed analysis of the surface charge in PEMs is undertaken, a comparison with the electrophoresis behavior of bare silica capillaries is useful. Wall charge on bare silica is controlled by silanol ionization.43 EOF is minimal below pH 4 and rises as the silanols are ionized at higher pH. The range of µeo is from 1 to 8 × 10-4 cm2 V-1 s-1. Interestingly, the maximum effective surface charge at the highest pHs, determined by electrophoretic measurements, is never more than 10% of the total number of available silanols.43 Turning to the question of surface charge in PEMs, we will make use of arguments presented by Donath et al.44 in their electrophoretic analysis of polyelectrolyte-coated latex particles. A system comprised of multilayer-coated particles moving through aqueous buffer is symmetrically inverted to the system studied here. There are many interrelated effects that conspire to make the electrophoretic behavior of a surface coated with a porous “hairy” layer substantially different from that of a smooth, impenetrable surface. The surface charge is characterized by an apparent charge density, σapp, which is obtained from eq 2 by inserting experimental µeo and κ values (reflective of the buffer composition). Here, σapp will always be less than σsurf, and their relative magnitudes is an indication of how much deviation exists between experiment and the ideal Helmholtz/Smoluchowski behavior of a smooth, impenetrable surface with low charge (where σapp ) σsurf). Factors Influencing the Effective Surface Charge. First, interior charges within a thin film of polymer will be shielded by those at the surface.44 If the Debye length is small (high ionic strength), most interior charged groups are screened and do not contribute to the EOF outside the film. Under our conditions (e.g., pH 4, 20 mM phosphate buffer) the Debye length is 4.6 Å. Second, when the more complete (nonlinear) form of the PoissonBoltzmann equation is employed (i.e., beyond the low potentials where the linear Debye-Hu¨ckel approximation is operative), σeff decreases substantially, particularly at high σsurf. Third, if thick and highly charged layers are employed, it is necessary to include surface conductivity, that is, the transport of ions on and through the surface layer. The consequence of surface conductivity is to reduce σeff, especially at low buffer concentrations. Finally, if the electrophoretic flow can penetrate the charged hairy polymer layer, the apparent charge density, and thus EOF, is significantly enhanced. An additional effect will be considered here. Due to the high charge density on polymer chains and the cylindrical geometry of a polymer chain, a strong electric field traps counterions near the polymer (“counterion condensation”) and the effective charge on the polymer backbone approaches a limiting value.45 For water, (43) Mosher, R. A.; Zhang, C.-X.; Caslavska, J.; Thormann, W. J. Chromatog. A 1995, 716, 17. (44) Donath, E.; Walther, D.; Shilov, V. N.; Knippel, E.; Budde, A.; Lowack, K.; Helm, C. A.; Mo ¨hwald, H. Langmuir 1997, 13, 5294. (45) Manning, G. S. J. Chem. Phys. 1969, 51, 924.

Figure 4. Electroosmotic mobility (magnitude) of bare silica ([), 6.5-layer pair cationic coating (9), and 7-layer pair anionic coating (2) at different phosphate concentrations (ln mM). The negative coating and positive coating were deposited with the same procedures as the coatings used for Figures 5 and 6, respectively. Phosphate pH was held constant at 6.0, 37-cm length capillary (30 cm to detector), 15-kV operating voltage, electrokinetic injection, 254 nm detection, and acetone used as EOF marker.

Figure 5. Electroosmotic mobility dependence on pH for a 7-layer pair multilayer coating (negatively charged). The capillary used in Figure 6 was employed for this experiment with an additional layer of PSS deposited with 0.5 M NaCl. The running electrolyte was 20 mM phosphate, 37-cm length capillary (30 cm to detector), 15-kV operating voltage, electrokinetic injection, 254 nm detection, and acetone used as neutral marker. No equilibration time was needed. Even after large excursions in pH, the EOF immediately adopts a stable and reproducible value.

this limiting value corresponds to one charge per 0.7 nm (the Bjerrum length). Donath et al. found it necessary to invoke counterion adsorption to explain satisfactorily their electrophoresis results.44 Although this was not termed “condensation” of counterions, the effect would be the same. The impact of these effects to yield a substantial decrease in σsurf can be illustrated by comparing σsurf and σeff. The latter was in the range of 0.02 ( 0.01 C m-2 at pH 4-6 for anionic or cationic outer layers and 20 mM phosphate. This is between 0.7 and 3% of σsurf (eq 4) for the layers prepared from 1 to 0.1 M salt in the respective deposition solutions. It is of interest to identify the principal contributors to the decrease in “available” surface charge. Since deposition from 1 M salt yields the equivalent of 10 dry monolayers of excess polymer charge, it is clear that much of the charge is buried, screened by layers nearer the film/solution interface. A study into the effect on µeo of ionic strength and σsurf (electrophoretic fingerprinting)46 would shed light on which other mechanisms chiefly limit the effective surface charge. Preliminary measurements in this direction have been made. For example, µeo is roughly constant with increasing buffer concentration (Figure 4), which would imply, since κ increases (eq 3), an increasing σeff. This is possible if significant layer flow penetrability is invoked.44 Electroosmotic flow inside the hairy layer is less subject to double layer compression with increasing ionic strength. For comparison, the ionic strength dependence of µeo of bare silica is also depicted in Figure 4. In the (reasonably high) range of ionic strength surface conductivity probably plays a lesser role in limiting σeff. pH Dependence of PEM-Coated Capillaries. In electrophoretic studies of particles the surface is often composed of weak acids or bases. σsurf is thus controlled by buffer pH. In their study on PEMs on carboxylate-coated latex particles, Donath et al.

clearly observed the pH dependence of the underlying substrate,44 which represented a substantial fraction of σsurf. The differences with the PSS/PDADMAC system used here are several: the polymers are fully ionized at all experimental pH values, and the layers are much thicker. It is unlikely that the silanol ionization is directly “viewed” through a 450-Å thick multilayer. It is entirely possible that the excess silanol charge can be transmitted to the PEM surface via a series of low-amplitude polymer reconformations. This would represent, however, a small change in σsurf, with correspondingly low changes in µeo. For example, for a PEM deposited from 1 M salt, the silanol coverage can add a maximum of -0.06 C m-2 (4% of σsurf) for a pH change of 4 to 8. Although the mobility of a negatively charged PEM is, as expected, only weakly dependent on pH (Figure 5), the positively charged columns exhibited more pronounced pH dependence (Figure 6). This difference cannot be ascribed to the relatively small changes in σsurf from silanol groups. We believe it is due to adsorption. At higher pH, a greater fraction of the phosphate exists in the HPO42and PO43- forms, which, being highly charged, would condense (adsorb) more strongly onto the positive surface. Column Performance: Reproducibility, Conditioning, and pH Extremes. PEM-coated capillaries for capillary zone electrophoresis (PEM-CZE, if an acronym is sought) exhibit several desirable features for analytical separations. Run-to-run stability over a wide range of pH is evident in Figures 5 and 6. A stable µeo was established as soon as columns (wet or dry) were contacted by buffer, in contrast to the behavior of bare silica, where an “equilibration” period (up to two weeks) is typically required before the EOF stabilizes under given conditions.47 We attribute this to rapid solvation and swelling of the charged layer in PEMs when wetted.

(46) Marlow, B. J.; Rowell, R. L. Langmuir 1990, 6, 1088.

(47) Lambert, W. J.; Middleton, D. L. Anal. Chem. 1990, 62, 1585.

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Table 2. Migration Times and Theoretical Plate Numbers from Separation of Four Basic Proteins with 6.5-Layer Pair PDADMAC/PSS Multilayer Coating

protein R-chymotrypsinogen A ribonuclease A cytochrome c lysozyme

Figure 6. Electroosmotic mobility dependence on pH for a 6.5-layer pair multilayer coating (positively charged) deposited with no salt for the first three, layer pairs and 0.5 M NaCl for the last three and a half layer pairs. Electrophoresis conditions as in Figure 5. No equilibration time was needed.

Capillary-to-capillary reproducibility was impressive. Several different capillaries were modified with 6.5-layer pairs.48 Of these, six were employed for reversed electroosmotic mobility studies at pH 6, and five were tested at pH 4. The µeo for the former was -2.69 × 10-4 cm2 V-1 s-1, with a relative standard deviation (RSD) of less than 2%, and the µeo of the latter group was -3.73 × 10-4 cm2 V-1 s-1, with a RSD of less than 2%. The constancy of EOF with respect to the layer thickness (Figure 3) is, no doubt, a contributing factor to reproducibility. Exposure to extremes of pH, particularly basic conditions, can strongly perturb bare silica or chemically bonded silica. Here, a positively charged multilayer (6.5-layer pairs) was constructed and four consecutive runs with 20 mM phosphate, pH 4.0, yielded a µeo of -3.79 × 10-4 cm2 V-1 s-1 for each run. The capillary was then flushed with 0.01 M NaOH (pH 12) for 10 min and rinsed for 5 min in the pH 4.0 phosphate buffer. Three consecutive electrophoresis runs resulted in an average mobility of (-3.76 ( 0.02) × 10-4 cm2 V-1 s-1. The rinsing in strong base was repeated, and three more separations were completed with a µeo of (-3.78 ( 0.02) × 10-4 cm2 V-1 s-1. The same capillary was later flushed with 0.01 M HCl (pH 2) for 10 min and then rinsed with pH 4.0 phosphate. Three consecutive runs yielded a µeo for each of -3.79 × 10-4 cm2 V-1 s-1. The acid rinse was repeated, and the mobility for all three runs was again -3.79 × 10-4 cm2 V-1 s-1. The durability of the multilayer coating is in accord with the generally observed stability of PEMs in conditions of extreme pH and/or ionic strength.30,39 The long-term reproducibility of a PEM capillary was then investigated. A 6.5-layer pair multilayer was constructed, and nearly 100 separations, including 20 separations of the basic protein combination (Tables 1 and 2), were performed for 5 of the next 6 days. Each separation (“run”) was 10-20 min in duration with applied voltages of 10 or 15 kV. Separations were done at pH 4.0 and 6.0 with running electrolyte concentrations as (48) Multilayers with the first few monolayers deposited from salt-free solutions were more stable.

4012 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

migration theoretical migration theoretical times (min) plates (N) times (min) plates (N) at pH 4.0 at pH 4.0 at pH 6.0 at pH 6.0 5.56 ( .08 6.01 ( .08 7.94 ( .12 8.72 ( .07

131 000 157 000 241 000 216 000

6.09 ( .02 6.37 ( .01 8.67 ( .03 11.46 ( .04

155 000 157 000 236 000 179 000

high as 100 mM. No changes in electroosmotic mobility were observed during these 6 days. On the sixth day, an additional layer of PSS was adsorbed onto the multilayer. Over the next 2 days, 22 runs were done in the pH range 4.0-8.0. During this time, no significant changes in µeo at different pH were observed in comparison to values presented in Figure 5 (e.g., 4.17 × 10-4 cm2 V-1 s-1 at pH 6). After these experiments, the capillary was removed from the instrument and dried out over a 2-week period. The capillary was placed back in the instrument, and five electrophoresis runs were performed with pH 6.0, 20 mM phosphate. The µeo was still 4.21 × 10-4 cm2 V-1 s-1 for each run as compared to 4.17 × 10-4 cm2 V-1 s-1 from 2 weeks earlier. The capillary was again removed from the instrument and dried for another 34 days. When the capillary was placed back into the instrument, the neutral marker was measured at an average mobility of 4.23 × 10-4 cm2 V-1 s-1 for four runs. The reproducibility of protein migration times was in the range of 1-1.5% (Table 2). The reproducibility of migration times of the negative (2-PPA) and neutral marker was within 0.5% for replicate analyses on the same day and within 1.5% for replicate analyses over a period of 2 weeks interspersed with other samples. Separation of Proteins. As discussed above, a single adsorbed layer of positive polyelectrolyte proved reasonably effective in promoting reversed flow CZE separations of basic proteins (Figure 1). For separations at pH 6, this column had to be “refreshed” by rinsing with polyelectrolyte between runs. Also, some tailing was still observed. PEM-coated capillaries proved particularly effective in separating the proteins. Figure 7 depicts an electropherogram employing silica coated with 6.5-layer pairs of PDADMAC/PSS. Elution order and time are comparable to the single-layer PDADMAC column (Figure 1), but the resolution has improved. Efficiencies of protein separation using the PEM column at pH 4 and 6 are summarized in Table 2. Although additional efforts to optimize the injection technique were not made, it is unlikely that injection technique was the limiting factor in efficiency, given that there was no trend in retention time vs efficiency. It is feasible that small capacity factors (for example, slight partitioning of hydrophobic areas of protein into the PEM) limit efficiency. Protein adsorption is largely suppressed. Electrostatic repulsion of like charges is an obvious mechanism for inhibition of protein adsorption on both the monolayer and multilayer columns. In the former, underlying silanols may not have been rendered completely inaccessible to proteins, allowing residual electrostatic, hydrogen bonding, or hydrophobic interactions. On the other hand, it is feasible that Coulombic interactions are not principally responsible for reduced protein adsorption. It is possible, for

Figure 7. Separation with 6.5-layer pair multilayer coating (first three layer pairs with no salt polyelectrolyte solutions, last three and a half with 0.5 M NaCl) at pH 4.0 of four basic proteins used in Figure 1. Fifteen kV applied voltage. Other separation conditions as in Figure 1.

example, for polymers to adsorb on like-charged surfaces under certain conditions.49 Mechanisms that would promote adsorption include interactions between hydrophobic areas of protein and the hydrophobic backbone of the polyelectrolyte.4 Also, recent studies into the charge distribution within PEMs have revealed that both negative and positive polyelectrolyte charges can coexist at the surface, at least in salt-containing solution.50 If this were the case, localized Coulombic attractions would be possible. Countering these attractive interactions are the loss of configurational and translational entropy typically considered in classical macromolecule adsorption theory,51 as well as excluded volume arguments. Specifically, an entropy penalty is paid if the free volume of polyelectrolyte segments is reduced by invading protein. Since the PEM surface is probably very hydrated, the overall adsorption enthalpy for a protein sticking to what is essentially a layer of water will be low. Capacity Factors for Thicker Films: Separation of Neutral Solutes. The high efficiencies for protein separations provide an additional argument for minimal interaction of proteins with the PEM: even small capacity factors have been predicted to reduce efficiency significantly.3 We have, however, noticed that some of the smaller analytes appear to partition into the PEM film, particularly for thick films. Partitioning would be useful in separating neutral species. Figure 8 shows the separation of four neutral species: acetone,52 fluorobenzene, phenol, and p-cresol using a 6.5-layer pair PDADMAC/PSS column built from solutions containing 0.5 M salt and 30% ethanol. The addition of organic modifiers during PEM buildup has been shown to enhance film (49) Cosgrove, T.; Obey, T. M.; Vincent, B. J. Colloid Interface Sci. 1986, 111, 409. (50) Lowack, K.; Helm, C. A. Macromolecules 1998, 31, 823. (51) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman and Hall: London, 1993. (52) Acetone as EOF marker was not retained by partitioning into the multilayer since it coelutes with D2O. (53) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163.

Figure 8. Separation of (1) acetone, (2) fluorobenzene, (3) phenol, (4) p-cresol with 6.5-layer pair PEM. Polyelectrolytes deposited from 0.5 M NaCl in 30% ethanol/water to provide a film of ∼200 nm thickness. Separations conditions as in Figure 2. Table 3. Number of Theoretical Plates and Capacity Factors for Acetone and Three Neutral Benzene Derivativesa solute

theoretical plates (N)

capacity factor (k′)

acetone fluorobenzene phenol p-cresol

50700 21600 9200 4300

0.0 0.12 0.35 0.47

a

See Figure 8.

thickness.31 The approximate thickness of this multilayer made from 30% ethanol was 200 nm. Table 3 summarizes the efficiencies and capacity factors for these neutral compounds, supporting the conclusion3 that partitioning into the multilayer comes at the expense of efficiency. CONCLUSIONS The PEM-coated columns described herein represent a fruitful intersection of materials and separations science. The facility with which these stable columns are prepared, coupled with the compositional flexibility of multilayer-containing thin films, suggests versatility as a phase for CZE. Whereas the work reported above has been limited to multilayers of polyelectrolytes only, many other charged species, such as biopolymers or colloidal particles, may be incorporated within PEMs to enhance potential partitioning-driven separations. Interestingly, enzymes retain their activity within PEMs,53 suggesting biospecific interactions for separations may be built into these systems. In this respect, the absence of irreversible protein adsorption is particularly encouraging. ACKNOWLEDGMENT This work was supported by the National Science Foundation (Grant DMR 9727717). We are grateful to John Dorsey for helpful discussion and for providing us with access to a CE instrument. Received for review March 15, 1999. Accepted July 11, 1999. AC990277L Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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