Synthesis and Characterization of Capillary Columns Coated with

Anal. Chem. 1996, 68, 2731-2736

Synthesis and Characterization of Capillary Columns Coated with Glycoside-Bearing Polymer Marcella Chiari,* Norma Dell’Orto, and Arianna Gelain

Istituto di Chimica degli Ormoni, Via Mario Bianco 9, 20131 Milano, Italy

Fused silica capillary columns were coated with a polymer obtained by radical polymerization of a novel acrylamido derivative, [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose (AEG). This monomer, bearing a monosaccharide residue, was synthesized by direct coupling of [(Nacryloylamino)ethoxy]ethanol with glucose. The aim of this work was to demonstrate that weak, reversible interactions that take place between wall polymer coatings and protein analytes lead to poor reproducibility of transit times and capillary performance degradation. The pronounced hydrophilicity of the new poly(AEG) phase improves the reproducibility of transit times and the peak efficiency, as demonstrated by a complete series of tests carried out at various pH values. Capillary zone electrophoresis (CZE), with its high-resolution power, mass sensitivity, and quantitative capability, is becoming an important tool for the separation and quantification of biopolymers. In particular, in the analysis of proteins, CE offers a method complementary to liquid chromatography and potentially an alternative to slab gel techniques. When the electrophoretic process is carried out in a capillary with an internal diameter of 25-100 µm, the Joule heat is dissipated from the surface more efficiently than from a slab gel, allowing the use of higher voltage and increasing separation efficiency and velocity. In theory,1,2 one could expect to achieve separation efficiencies of millions of theoretical plates in CE of proteins, since a field strength as high as 30 kV can be employed in the separation, and the protein diffusion coefficient is reasonably low. In reality, the efficiency typically achieved in protein separation is considerably lower. Protein peaks are often broad and in several cases not visible at all, especially when the proteins are analyzed below their isoelectric point. Such a large reduction in efficiency is due to interactions between proteins and the silica wall. Fused silica surfaces are characterized by immobilized charges responsible for electrostatic interactions with protein samples. Reversible interactions between analytes and the capillary surface worsen the separation profile, broadening the peaks and decreasing reproducibility, while irreversible interactions completely destroy the profile. Various methods have been used to diminish or control the effects of silanols on protein separation. In addition to approaches that modify the properties of silica by acting on pH or on BGE composition3-13 EOF and adsorption have been controlled through dynamic14,15 or covalent modifications of the fused silica surface.16-20 (1) Jorgenson, J. W.; Lukacs K. D. Anal. Chem. 1981, 53, 1298-1302. (2) Hjerte´n, S. J. Chromatogr. 1985, 347, 191-198. (3) McCormick, R. M. Anal. Chem. 1988, 60, 2322-2328. (4) Lauer, H. H.; McManigill, D. Anal. Chem. 1986, 58, 166-170. (5) Walbroehl, Y.; Jorgenson, J. W. J. Microcolumn Sep. 1989, 1, 41-45. S0003-2700(96)00158-8 CCC: $12.00

© 1996 American Chemical Society

Although more laborious, methods that shield surface silanol groups by covalently attaching a neutral polymer to the wall appear to be the most attractive from the point of view of flexibility in the choice of separation conditions. We have recently described poly([(acryloylamino)ethoxy]ethanol) (poly(AAEE))-coated capillaries.21 In this type of coating a stable and hydrophilic acrylamido derivative, the [(N-acryloylamino)ethoxy]ethanol, forms linear chains that copolymerize with γ-methacryloxypropyl functions introduced onto the silica surface by catalytic hydrosilylation of allyl methacrylate on an SiH-modified fused silica capillary.22 The type of binding between the silica and the polymer and the characteristics of the polymer make these capillaries highly resistant to hydrolytic cleavage. In a previous study, we compared efficiency and transit time reproducibility in polyacrylamide- and poly(AAEE)-coated capillaries and evaluated their lifetimes.21 Our results unequivocally indicated the importance of polymer hydrophilicity and hydrolytic stability in providing suitable performance. The high chemical stability of poly(AAEE) leads to the production of columns with a longer lifetime, as demonstrated by long-term stability tests performed at alkaline pH. However, in the course of that investigation, we noted the presence of weak, reversible interactions with the capillary surface that led to poor reproducibility of the separation pattern. Therefore, in order to maintain adequate performance of the capillary over time, we proposed to treat the ends with a solution of methylcellulose (MC) every 15 h of continuous use. The rationale for this treatment was to provide (6) Lindner, H.; Helliger, W.; Dirschlmayer, A.; Jaquemar, M.; Puschendorf, B. Biochem. J. 1992, 283, 467. (7) Bushey, M. M.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 301-310. (8) Chen, F. A.; Kelly, L.; Palmieri, R.; Biehler, R.; Schwartz, H. J. Liq. Chromatogr. 1992, 15, 1143-1161. (9) Gordon, M. J.; Lee, K. J.; Arias, A. A.; Zare, R. N. Anal. Chem. 1991, 63, 69-72. (10) Zhu, M.; Rodriguez, R.; Hansen, D.; Wehr, T. J. Chromatogr. 1990, 516, 123-131. (11) Emmer, A.; Jansson, M.; Roeraade, J. J. High Resolut. Chromatogr. 1991, 14, 738-740. (12) Strege, M. A.; Lagu, A. L. J. Chromatogr. 1993, 630, 337-344. (13) Muijselaar, W. G. H. M.; De Bruijn, C. H. M. M.; Everaerts, F. M. J. Chromatogr. 1992, 605, 115-123. (14) Wiktorowicz, J. E.; Colburn, J. C. Electrophoresis 1990, 11, 769-773. (15) Tsuji, K.; Little, R. J. J. Chromatogr. 1992, 594, 317-324. (16) Ganzler, K.; Greve, K. S.; Cohen, A. S.; Karger, B. L. Anal. Chem. 1992, 64, 2665-2671. (17) Cobb, K. A.; Dolnik, V.; Novotny, M. Anal. Chem. 1990, 62, 2478-2483. (18) Schmalzing, D.; Pigee, C. A.; Foret, F.; Carrilho, E.; Karger, B. L. J. Chromatogr., A 1993, 652, 149-159. (19) Bruin, G. J. M.; Chang, J. P.; Kuhlman, R. H.; Zegers, K.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1989, 471, 429-436. (20) Towns, J. K.; Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 599, 227-237. (21) Chiari, M.; Nesi, M.; Sandoval, J. E.; Pesek, J. J. Chromatogr. 1995 , 717, 1-13. (22) Montes, M. C.; Van Amen, C.; Pesek, J. J.; Sandoval, J. E. J. Chromatogr. 1994, 688, 31-45.

Analytical Chemistry, Vol. 68, No. 17, September 1, 1996 2731

a dynamic coating to a region of the capillary that is repeatedly dipped into a highly concentrated protein solution. Neutral linear polymers such as MC shield proteins that are eventually adsorbed onto the wall and counteract the effects of wall-immobilized charges. Proteins interact reversibly or irreversibly with a surface in several ways, one being electrostatic interactions with unshielded silanol,23 and another being hydrophobic interactions between hydrophobic patches and polymer hydrophobic reagions.24 To investigate the effects of interactions between analytes and polymer coatings, the separation of test proteins in columns coated with various acrylamido polymers ranging from hydrophobic to very hydrophilic was systematically evaluated. The aim of this work was to see whether or not hydrophilic acrylamido coatings would provide improved peak shapes and reproducibility of migration time. We proposed to suppress protein-wall interactions by coating capillary columns with a novel N-substituted polyacrylamide. By direct coupling of [(N-acryloylamino)ethoxy]ethanol with glucose, we synthesized a new highly hydrophilic acrylic monomer, [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose (AEG). Acrylic monomers bearing monosaccharide residues that have a glycoside structure linked with a reactive acrylate, referred to as glycoside monomers, have been recently used to produce polymers with an artificial backbone and pendant saccharide units.25 One field of research into these polymers concerns their use as surface or polymer modifiers.26,27 We proposed to coat the surface of capillaries previously activated with allyl double bonds with the glycoside polymer obtained by radical polymerization of AEG. Poly(AEG) suppresses the electoosmotic flow more efficiently than other hydrophilic natural polymers because the polymer built “in situ” from a monomer solution of appropriate concentration is more efficiently attached to the silica wall and properly shields wall silanols. In this paper, we present a complete evaluation of this type of capillary after investigating the efficiency and reproducibility of separations of test protein mixtures at different pH values over a long period of time. EXPERIMENTAL SECTION Reagents. Tris(hydroxymethyl)aminomethane (Tris), N,Nbis(2-hydroxyethyl)glycine (Bicine), R-chymotrypsinogen A, 3-{N[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS), 6-aminocaproic acid (EACA) β-lactoglobulin from bovine milk, trypsin from bovine pancreas, soybean trypsin inhibitor, myoglobin from horse skeletal muscle, trypsinogen from bovine pancreas, and R-lactalbumin from bovine milk were purchased from Sigma Chemical Co. (St. Louis, MO). Lysozyme from hen egg white and ribonuclease A from bovine pancreas were from BoehringerMannheim (Mannheim, Germany). Acrylamide, ammonium peroxydisulfate, and N,N,N′,N′-tetramethylenediamine (TEMED) were from Bio-Rad Labs (Hercules, CA). β-D-Glucose pentaacetate, mercuric bromide, and N,N-dimethylacrylamide (DMA) were from Aldrich Chemie (Steinheim, Germany). Toluene, methyl alcohol, ethyl acetate, petroleum (23) Towns, J. K.; Regnier, F. E. Anal. Chem. 1992, 64, 2473-2478. (24) Zhao, Z.; Maik, A.; Lee, M. L. Anal Chem. 1993, 65, 2747-2752. (25) Kitazawa, S.; Okamura, M.; Kinomura, K.; Sakakibara, T. Chem. Lett. 1990, 9, 1733-1736. (26) Kimura, S.; Hirai, K. Macromol. Chem. 1962, 58, 232. (27) Schweiger, R. G. J. Polym. Sci. 1964, A2, 2471-2479.

2732 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

Figure 1. Synthesis of [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose (AEG).

ether, silica gel 60F254, silica gel 60 (230-400 mesh), and mercuric oxide were from Merck (Darmstadt, Germany). Amberlite IRA 400 and 2-amino-2-methyl-1,3-propanediol (AMPD) were from Fluka (Buchs, Switzerland). Water and all other reagents were of analytical grade. (N-Acryloylamino)ethoxy]ethanol (AAEE) was synthesized as described by Chiari et al.28 Apparatus. 1H NMR spectra at 500.13 MHz were acquired on a Bruker DMX500 spectrometer. The FABMS spectrum was obtained using Xe as a gas on a VG-7070-EQ-HF instrument equiped with its own source. The IR spectrum, in KBr, was measured with an Isco FT-IR 5000 spectrometer. Sixteen interferograms were collected with a resolution of 4 cm-1. CZE separations were performed in a Waters Quanta 4000 capillary electrophoresis system (Millipore, Milford, MA) and in a SpectraPhoresis 1000 capillary system (Thermo Separation Products, Freemont, CA). Data were collected on a personal computer using SW-Phoresis 1000 software. In all the experiments, 75 and 50 µm fused silica capillaries (Polymicro Technologies, Phoenix, AZ) coated as described below were used. Proteins and benzyl alcohol, used as a marker of electroosmosis, were detected at 214 nm. Protein stock solutions (10 mg/mL) were prepared in water and diluted to the final concentration just before the injection. Between runs, the column was rinsed with the separation buffer by hydrodynamic pressure for 2 min. EOF was measured at 600 V/cm in 25 mM Bicine-Tris buffer, pH 8.5. The samples were electrokinetically injected by applying 15 kV for 0.5 s. Synthesis of [(N-Acryloylamino)ethoxy]ethyl-β-D-glucopyranose (AEG, 2). Figure 1 depicts the synthesis of AEG. To [(N-acryloylamino)ethoxy]ethanol was added under nitrogen 1.64 g (0.013 mol) of R-D-bromopentacetylglucose, dissolved in 35 mL of anhydrous methylene chloride. The solution contained 1.99 g (9.2 × 10-3 mol) of mercuric oxide and 0.165 g (4.5 × 10-5 mol) of mercuric bromide as catalysts and 0.097 g (5.8 × 10-4 mol) of 4-tert-butylcatechol as inhibitor. The solution was stirred at room temperature (20 h) with frequent monitoring by TLC on silica gel (ethyl acetate/petroleum ether 8:2). When the TLC pattern no longer changed, the reaction mixture was recoverd by filtration (28) Chiari, M.; Micheletti, C.; Nesi, M.; Fazio, M.; Righetti, P. G. Electrophoresis 1994, 15, 177-186.

and evaporated to a syrup. The crude product was purified by silica gel flash column chromatography, using a mixture of CH3COOEt/petroleum ether (95:5) as eluent, to give 0.8 g (1.2 × 10-3 mol) of pure 2 (15% yield). For the hydrolysis of acetylglucopyranose (3), 0.59 g (0.0012 mol) of 2 dissolved in 10 mL of methanol was stirred for 2 h in the presence of a suspension of 3.9 g of Amberlite IRA 400 resin. After the reaction was completed, the resin was filtered off and washed with methanol, and the filtrate and washings were combined and evaporated to give 0.335 g of 3 (85% yield), pure by TLC (CH3COOEt, CH3OH, H2O, 9:3:1). 1H-NMR (DMSO): δ 8.25 (1H, br t, NH), 6.35 (1H, dd, Ha), 6.18 (1H, dd, Hb), 5.78 (1H, dd, Hc), 4.25 (1H, d, H1), 3.98 (1H, m), 3.9-3 (12H, m). FABMS: m/z 341 (M + Na)+, 319 (M + H)+, 192, 181, 159, 97. IR: 3366 cm-1, ν(OH, NH), 1659 cm-1 (amide I), 1626 cm-1, ν(CdC), 1551 cm-1 (amide II), 1100 cm-1 ν(C-O alcohol), 1050 ν (C-O ether). Alkaline Hydrolysis of Free Monomers. A 20 mM solution of monomer in 0.1 M NaOH or in 0.1 M HCl was incubated at 70 °C for up to 6 h. At given time intervals, aliquots were collected and diluted (to ∼1 mM) in 0.1 M sodium borate buffer, pH 9.0, containing cetyltrimethylammonium bromide (CTAB) (20 mM). Immobiline, pK 9.3 (0.5 mM), was added as internal standard. The samples were analyzed by CZE in an uncoated capillary, 75 µm i.d., 42 cm long, using 0.1 M sodium borate, pH 9.0, containing 20 mM CTAB as running buffer. A potential of 8 kV was applied, and then the samples were injected by hydrodinamic pressure for 1 s at the cathodic end and detected by a UV detector set at 254 nm. The sample concentration was calculated from the peak area using a calibration curve. The decrement of the concentration was used to assess degradation of the monomer. Coating Procedure. Capillaries coated with (1) poly(dimethylacrylamide), (2) polyacrylamide, (3) poly(AAEE), and (4) poly(AEG) were prepared as follows. The four polymers were covalently linked to surfaces activated with γ-methacryloxypropyl functionalities bonded to the wall through a direct silicon-carbon linkage, prepared as described in ref 22. Briefly, a hydridemodified support was formed by reacting triethoxysilane (TES) with a silica substrate in the presence of water and hydrochloric acid, using dioxane as the solvent. The γ-methacryloxypropyl group residue was linked to the hydride-modified substrate by hydrosilylation of allyl methacrylate in the presence of a Pt catalyst. γ-Methacryloxypropyl-activated capillaries were filled with solutions of dimethylacrylamide (3.8% v/v), acrylamide (3% w/v), [(N-acryloylamino)ethoxy]ethanol (6% v/v) and [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose (15% w/v). The four monomer solutions were degassed under vacuum (20 mmHg) for 40 min, and the appropriate amounts of catalyst and initiator (1 µL of TEMED and 1 µL of 40% ammonium peroxydisulfate per milliliter of gelling solution) were added. Polymerization was allowed to proceed overnight at room temperature. The capillaries were then emptied with a syringe or an HPLC pump, according to the viscosity of the polymer solution. Separation Conditions. Alkaline proteins (lysozyme, ribonuclease A, R-chymotrypsinogen A, and myoglobin) were separated at in 20 mM 6-aminocaproic acid titrated to pH 4.4 with acetic acid (EACA-HAc) at 528 V/cm (8 µA); the polarity of the power supply was positive (anodic injection). Acidic proteins, trypsin inhibitor, β-lactoglobulins A and B, and R-lactalbumin, and L-asparaginase were resolved with 25 mM

Figure 2. Kinetics of hydrolysis of different monomers in free solution. Conditions: 0.1 N NaOH, 70 °C, for 6 h. The percentage of undegraded monomer was estimated by CZE. Abbreviations: DMA, dimethylacrylamide; AAEE, [(N-acryloylamino)ethoxy]ethanol; AEG, [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose.

Bicine-Tris, pH 8.5, or 20 mM TAPS-AMPD, pH 8.8, as buffers. A field of 500 V/cm was applied. The polarity of the power supply was negative (cathodic injection). RESULTS AND DISCUSSION Characteristics of AEG Monomer. A novel acrylic monomer bearing a monosaccharide residue was synthesized by direct coupling of [(N-acryloylamino)ethoxy]ethanol with glucose. The monomer was separated from the reaction mixture by silica gel column chromatography and identified by NMR, IR, and mass spectroscopies. A number of studies have indicated that glycoside monomers are very safe materials that are easily polymerized to yield homopolymers and copolymers with characteristics suitable for applications in several fields.25,26 It has also been shown that only a small number of saccharide moieties are needed to make copolymers highly cohesive and hydrophilic and that it is possible to make hydrophilic surfaces from hydrophobic polymers by introducing saccharide monomers. The aim of this current work was to suppress EOF and protein-wall interactions in capillary columns by coating the inner wall with a linear poly(AEG) polymer. Before being applied to the production of a novel type of polymeric coating, the novel monomer was subjected to a series of tests to determine its hydrolytic stability at alkaline and acidic pH values and its polymerization efficiency. The graph in Figure 2 shows the kinetics of hydrolysis for different N-substituted acrylamido monomers in mild alkaline solution (0.1 N NaOH, 70 °C). The behavior of the novel monomer AEG is intermediate between that of DMA and AAEE. It is well known that mono- and disubstituted acrylamides are more resistant to alkaline hydrolysis than unsubstituted species.27 However, some N-substituted acrylamido monomers are degraded very rapidly in alkaline solution.28 In general, the insertion of OH groups into the nitrogen alkyl substituents to increase the hydrophilicity may render the monomer extremely prone to hydrolysis. To prevent the formation of an ester by intramulecular rearrangement the OH groups should be more than three atoms away from the carbonyl group of the amido bond to preclude the formation of stable rings closing onto the carbonyl group.29 The molecular structure of AEG fulfills this requirement as the hydroxyl groups are 11 atoms away from the oxygen of the (29) Miertus, S.: Righetti, P. G.: Chiari, M. Electrophoresis 1994, 15, 1104111.

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2733

Figure 3. Schematic representation of poly(AEG) coating.

carbonyl, thus disfavoring hydrogen bonding. The insertion of an oxygen atom along the alkyl chain further increases the hydrophilicity of the molecule. The stability of glycoside bond in acidic solution, 0.1 N HCl, was also very good, and there was no degradation after 6 h of exposure of the monomer to a 0.1 N HCl solution at 70 °C. The stability of the monomer under both alkaline and acidic conditions is fully compatible with its potential use in electrophoretic applications, especially since the stability of the amido linkage increases when a monomer is incorporated into a polymer. Finally, we evaluated the efficiency of polymerization of the poly(AEG) homopolymer. Spectrophotometric measurements of the amounts of unreacted, conjugated acrylic double bonds, carried out at 254 nm, indicated a conversion greater than 99% of the monomer into the polymer. Poly(AEG) Coating Characterization. Figure 3 shows the structure of a poly(AEG) coating. Linear polymer chains are covalently attached to a surface previously activated by methacryloxypropyl functionalities. In a recent paper,21 we discussed the advantages of a two-step process in which silica is derivatized with allyl groups bonded to silanols through a stable Si-C linkage. In this previous work, we coated the inner surface with a novel N-substituted acrylamido monomer, [(N-acryloylamino)ethoxy]ethanol. This polymer, instead of polyacrylamide, increased the hydrolytic stability of the column. The quality of the poly(AAEE) coating was evaluated by monitoring the efficiency and the reproducibility of migration time in separation of alkaline and acidic proteins, and the results were compared with those obtained with classical polyacrylamide columns. A systematic investigation of the long-term stability evidenced that hydrolytic cleavage of polyacrylamide is not the main source of degradation of capillary performance.21 During electrophoresis in polymer-coated capillaries, there are weak interactions between proteins and the capillary wall, leading to retardation of transit time and broadening of the peak. To look into the nature of these interactions, we evaluated run-to-run reproducibility of migration time in capillaries coated with polyacrylamide polymers bearing different types of nitrogen substituents. Figure 4 shows four types of coatings based on dimethylacrylamide (DMA), acrylamide (AA), AAEE, and AEG. In the figure, the hydrophilicity of the four monomers is indicated by the logarithm of the octanol/water partition coefficient (log P). Log P was calculatated using atomic parameters according 2734 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

Figure 4. Reproducibility of migration times in columns with different polyacrylamide coatings. RSDs of retention times of 40 consecutive injections of (a) trypsin inhibitor, (b) β-lactoglobulin A, (c) β-lactoglobulin B, and (d) R-lactalbumin. The samples (0.1 mg/mL each) were separated in 25 mM Bicine-Tris pH 8.5 buffer at 500 V/cm. The hydrophilicity of the four acrylamido monomers, dimethylacrylamide (DMA), acrylamide (AA), [(N-acryloylamino)ethoxy]ethanol (AAEE), and [(N-acryloylamino)ethoxy]ethyl-β-D-glucopyranose (AEG) is indicated by log P, calculated from atomic parameters according to ref 29.

to Viswanadhan et al.30 An acidic protein test mixture containing trypsin inhibitor, β-lactoglobulins A and B, and R-lactalbumin was run in the four capillaries at pH 8.5 in 20 mM Bicine-Tris buffer. The conditions used in the test were very sensitive to the EOF effects. At high pH, Si-OH groups are deprotonated, and the EOF is maximized by the use of a buffer of low ionic strength. In (30) Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. N.; Robins, R. K. J. Chem. Inf. Comput. Sci. 1989, 29, 163-172.

Figure 5. Reproducibility of pattern profiles for 300 consecutive runs of acidic proteins in poly(AEG)-coated capillary: 1st, 150th, and 300th runs. Samples, from the left, are (1) trypsin inhibitor, (2) β-lactoglobulin A, (3) β-lactoglobulin B, (4) L-asparaginase, and (5) R-lactalbumin. Separation conditions: buffer, 20 mM Bicine-Tris at pH 8.5; capillary, 75 µm i.d. (37 cm total length capillary, 30 cm inlet to window); potential applied, 500 V/cm, reverse polarity; samples, injected electrokinetically for 1 s at 19 kV. Table 1. Reproducibility of Migration Timesa

addition, at this pH, low-pI proteins are negatively charged and thus repelled by the silanols. This reduces electrostatic interactions, leading to better evaluations of other kinds of interaction with the surface. The RSDs for the migration times of 40 consecutive injections, reported in Figure 4, indicate that run-torun reproducibility increases with the hydrophilicity of the surface. Proteins adsorbed to the inner wall produce effects on the peak profiles similar to those caused by the presence of unmasked silanols or, more generally, by the presence of immobilized charges. To counteract the effects of adsorbed proteins on reproducibility of time, we proposed, in a pervious paper, to dynamically coat the modified surface of the capillary with methylcellulose every 15 h of continous use.21 The success of this treatment in restoring the capillary performance showed that there are immobilized charges on the inner surface, while the relationship between the hydrophilicity of the polymer coating and the reproducibility of separation achieved indicates that hydrophobic interactions between protein patches and hydrophobic regions of the polymer are the most important factors in this. When the hydrophilic character of the polymer was increased, as in the case of poly(AEG), there was dramatic improvement in reproducibility. Table 1 summarizes the results of a stability test performed at pH 8.5 in 25 mM Bicine-tris buffer. The protein test mixture was run in a capillary of 75 µm i.d. The change in migration times after 300 consecutive injections was negligible, and the RSDs) for the migration times were still very low. Figure 5 shows the profiles of three representative protein electropherograms: the first, the 150th, and the 300th. The electrolyte buffer was renovated before each run, and the protein mixture was freshly prepared from stock solutions every 15 h. The capillary was used over a period of 100 h, and no reconditioning treatments were needed during the entire test period. When not in use, the capillary was kept in a 0.1% sodium azide solution in order to prevent microbial degradation of the polymer, as it is well known that bacteria might digest glycoside units attached to the polymer. Excellent batch-to-batch reproducibility was confirmed by the results achieved with three poly(AEG)-coated

protein

average time (min)

soybean trypsin inhibitor

4.64c

β-lactoglobulin A β-lactoglobulin B R-lactalbumin

4.67d 4.92 4.91 5.27 5.26 7.70 7.75

RSDb (%) 0.51c 0.68d 0.53 1.08 0.54 1.05 0.73 1.3

a Conditions: 25 mM Bicine-Tris pH 8.5 buffer; 500 V/cm. b Relative standard deviation. c n ) 40 (runs 1-40). d n ) 40 (runs 260-300).

Figure 6. Electropherogram of alkaline proteins (1) lysozyme, (2) ribonuclease A, (3) myoglobin, and (4) R chymotripsinogen A, carried out in the capillary used in the stability test of Figure 4. Separation conditions: buffer, 20 mM EACA-HAc pH 4.4; applied field, 538 V/cm; sample proteins (0.1 mg/mL), injected electrokinetically for 1 s at 21 kV at the anodic side.

capillaries batches, each consisting of three columns. The columns were evaluated with the basic and the acidic test protein mixtures separated as described in Figures 6 and 7, respectively. The RSDs for the migration times of the protein samples were