Anal. Chem. 2001, 73, 444-452
Chromatographic Interactions between Proteins and Sulfoalkylbetaine-Based Zwitterionic Copolymers in Fully Aqueous Low-Salt Buffers Camilla Viklund, Anna Sjo 1 gren, and Knut Irgum*
Department of Analytical Chemistry, Umeå University, S-901 87 Umeå, Sweden Ingolf Nes
Laboratory of Microbial Gene Technology, Agricultural University, N-1432 Ås, Norway
Macroporous monoliths containing N,N-dimethyl-Nmethacryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPE) have been synthesized via in situ photopolymerization, yielding a stoichiometric balance between sulfur and nitrogen in the final polymer, which is indicative of a genuine strong/strong zwitterionic character. The chromatographic properties of these zwitterionic resins were evaluated with respect to the retention behavior of inorganic ions and proteins. The weak electrostatic nature of the interaction between the sulfobetaine monoliths and proteins provided a high selectivity between basic proteins and peptides. Elution was accomplished with low-ionicstrength fully aqueous mobile phases, whereby high recovery was obtained, even for hydrophobic proteins. Chaotropic ions such as perchlorate or thiocyanate were used as mobile phase modifiers to modulate the apparent ion exchange group density, thus introducing a route for the modulation of the ionic strength that is required to competitively elute the protein. The promising features of polymeric sulfoalkylbetaine interaction layers for separation and analysis of biological extracts was also manifested in an application involving purification of biologically active peptide-pheromone obtained from Enterococcus faecium. The significance of bioseparations, both in industry and research, nurtures a perpetual search for novel stationary phases. Of particular interest are materials based on new separation modes that can provide selectivities that are complementary to existing techniques. Ion-exchange chromatography (IEC) is among the most widely used chromatographic tools in protein purification schemes, and is performed as both a high- and a low-pressure technique. Ion-exchange sorbents are usually based on inorganic silica particles or polymeric spheres, which have surface cationic or anionic groups that are capable of interacting electrostatically with ionic species of the opposite charge. The retention of a protein will, thus, depend on the charge status of the protein, the number of interaction sites available, and the strengths of the individual interactions. Displacement of proteinaceous material from the ion exchanger is normally achieved by competitively 444 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
increasing the ionic strength of the mobile phase, and relatively high salt concentrations are often required in order to elute even moderately retained proteins.1,2 Although many proteins can withstand high salt concentrations without severe denaturation or precipitation, there are usually other practical limits to the ionic strength of the eluent, such as the desire to obtain the separated components in a relatively low saline buffer in order to avoid extensive dialysis or desalting by gel filtration after the separation. Separation materials with dual charges have caught the interest of numerous experimenters in the field because of the conceptual advantage inherent in utilizing the amphoteric properties of both the separation materials and the analytes to model the retention of, for example, proteins. Experiments with multiple mode ion exchange chromatographic separations have been done with anion exchange and cation exchange columns connected in tandem3 or with mixed cation and anion exchange media using single columns packed with a mix of anion and cation exchange sorbents.4,5 The first time a separation material was furnished with separation moieties that could be termed zwitterionic was in 1981, when Knox and Jurand6 introduced a technique in which quadropolar ion pairs could be formed when 11-aminoundecanoic acid was added to the eluent to act as a “zwitterionpair agent”. This concept was used to alter the retention of various nucleotides and oligopeptides comprising up to three amino acids in a reversedphase chromatographic mode. Kurganov and co-workers7 were able to separate acidic and/or basic proteins using a stationary phase which contained both sulfonic acid and quaternary ammonium groups. This phase, which they called zwitterionic, although it should more appropriately be termed a mixed-mode phase, was prepared by introduction of sulfonic acid and quater(1) Karlsson, E.; Ryde´n, L.; Brewer, J. In Janson, J. C., Ryde´n, L., Eds. “Protein Purification: Principles, High-Resolution Methods, and Applications”, 2nd ed.; Wiley-Liss: New York, 1998; pp 145-205. (2) Chicz, R. M.; Regnier, F. E. Methods Enzymol. 1990, 182, 392-421. (3) el Rassi, Z.; Horva´th, Cs. J. Chromatogr. 1986, 359, 255-264. (4) Maa, Y. F.; Antia, F. D.; el Rassi, Z.; Horva´th, Cs. J. Chromatogr. 1988, 452, 331-345. (5) Freitag, R.; Splitt, H.; Reif, O. W. J. Chromatogr. A 1996, 728, 129-137. (6) Knox, J. H.; Jurand, J. J. Chromatogr. 1981, 203, 85-92;1981, 218, 341354; 1981, 218, 355-363; 1982, 234, 222-224. (7) Kurganov, A. A.; Davankov, V. A.; Unger, K. K. J. Chromatogr. 1991, 548, 207-214. 10.1021/ac000618r CCC: $20.00
© 2001 American Chemical Society Published on Web 12/22/2000
nary ammonium groups by sequential chloromethylation, sulfonation, and trimethylamination of a styrene layer superficially polymerized onto silica. The process resulted in ion exchange groups having different charges residing on separate phenyl moieties in the superficial layer. They noted two important aspects that clearly distinguish this mixed-mode phase from a true zwitterionic sorbent, that is, that the ion exchanger contained cationic groups in excess of anion exchange groups, and that the peak from lysozyme was broadened due to the mixed-mode interactions between the protein and the exchanger. The preparation of a bipolar HPLC stationary phase based on aminopropyl silica has been reported by Nomura et al.8 The amino groups were partially acylated with an acylating agent containing a C14 hydrocarbon chain attached through an ester bond. This ester bond was later hydrolyzed to form terminal carboxylic groups which, together with the original amino groups, resulted in anionic and cationic sites interspersed with hydrocarbon chains from unhydrolyzed ester. The material was used under reversed-phase conditions with high admixtures of organic solvents. The suitability of this quasi-zwitterionic sorbent for protein separation was explored, and it is apparent from their data that the separations took place by hydrophobic interactions, not by a solely ionic mechanism. The principle furthermore appears to have been abandoned, because some proteins were very strongly adsorbed and were, thus, difficult to elute. Tramposch et al.9 prepared a zwitterionic resin by reacting dimethylaminopropylsilane-modified silica with 1,3-propanesultone. They also showed that this stationary phase exhibits a weaker analyte adsorption, as compared to unmodified silica, when operated in the normal-phase chromatographic mode. Another zwitterionic stationary phase has been developed by Pidgeon and co-workers,10 who used columns containing immobilized phosphatidylcholine ligands to model the interactions between polar organic compounds with phospholipid membranes by calculating solute-membrane partition coefficients from chromatographic data. This chromatographic technique thus utilizes the experimental facileness of collecting chromatographic retention data with the more complicated task of imitating a biological system. Among the efforts to use multiply charged materials for separation, Yu et al.11,12 described the preparation of a chemically bonded zwitterionic silica sorbent and also showed its potential for the separation of nucleotides. However, no satisfactory results regarding separation of proteins were reported for this material. In the area of small-ion chromatography, Hu and co-workers conducted numerous studies13 in which commercial ODS columns were dynamically coated with zwitterionic surfactant reagents. On these coatings, simultaneous separation of inorganic cations and anions can be accomplished using pure water as the mobile phase. Our group also recently showed that methacrylate-based beads that were modified with covalently bonded zwitterionic groups are capable of similar interactions with inorganic ions.14 (8) Nomura, A.; Yamada, J.; Tsunoda, K. Anal. Chem. 1988, 60, 2509-2512. (9) Tramposch, W. G.; Weber, S. G. J. Chromatogr. 1990, 544, 113-123. (10) Ong, S. W.; Liu, H. L.; Pidgeon, C. J. J. Chromatogr. A 1996, 728, 113. (11) Yu, L. W.; Hartwick, R. A. J. Chromatogr. Sci. 1989, 27, 176-185. (12) Yu, L. W.; Floyd, T. R.; Hartwich, R. A. J. Chromatogr. Sci. 1986, 24, 177182. (13) Hu, W. Z.; Haddad, P. R. Trends Anal. Chem. 1998, 17, 73-79. (14) Jiang, W.; Irgum, K. Anal. Chem. 1999, 71, 333-344.
On summarizing this overview of chromatography in which opposite charges are used in the same separation, it can be concluded that practically all studies carried out so far with proteins have been based on a mixed mode in which the opposite charges are either physically separated from each other in separate interaction layers or are on particles of opposite charge that are packed in the same bed. In those cases where oppositely charged groups have been situated on the same carrier particle, functionalization has usually been accomplished by dynamic equilibration with amphiphilic zwitterions, or the covalently attached groups have been introduced in a sequential manner that disperses the charges randomly on the packing material without exact control over charge balance. When dynamic attachment has been used to impart zwitterionic functionality to hydrophobic separation materials, there is always a residual hydrophobic character that calls for reversed-phase conditions in the elution process. Linear polymers with zwitterionic side chains have been extensively studied in polymer physical chemistry due to the fascinating rheological properties emanating from their antipolyelectrolytic behavior.15-18 The most intensively studied class of zwitterionic polymers is composed of carboxyalkyl- or sulfoalkylbetaines, in which the cationic functionality (a quaternary ammonium group) and the anionic moiety (a terminal sulfonate or carboxylate group) are incorporated in close proximity, pendant to the polymer backbone. The sulfoalkylbetaine groups are particularly intriguing for separation purposes because of their pH-independent zero net charge. In a recent paper, we discussed the polymer chemistry aspects of the interactions between proteins and sulfoalkylbetaine zwitterions and demonstrated that the strength of these interactions is controlled by ionic strength and can be modulated by the presence of chaotropic agents.19 In this paper, we demonstrate that the concept of in situ formation of macroporous monolithic rods20 can be used with sulfoalkylbetaine methacrylate monomers for the direct polymerization of zwitterionic separation columns which are useful for separating basic proteins and peptides in fully aqueous buffers of low ionic strength. EXPERIMENTAL SECTION Reagents and Solutions. The zwitterionic monomer N,Ndimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE) was a gift from Raschig GmbH, Ludwigshafen, Germany, and was used without further purification. Ethylene dimethacrylate (EDMA, 90%) was purchased from Fluka, Buchs, Switzerland; triethylene glycol dimethacrylate (TEGDMA, 95%) and benzoin methyl ether (99%) were obtained from Aldrich, Steinheim, Germany. The methanol was of analyzed HPLC grade (J.T Baker, Deventer, Holland), and the water was purified by Milli-Q equipment (Millipore, Bedford, MA). Proteins used as test solutes were purchased from Sigma (St. Louis, MO) and were the following (CAS number; Sigma product number): R-chymotrypsinogen A type II, from bovine pancreas (9035-75-0; C4879); cytochrome c, from horse heart (9007-43(15) Hart, R.; Timmerman, D. J. Polym. Sci. 1958, 28, 638-640. (16) Monroy-Soto, V. M.; Galin, J. C. Polymer 1984, 25, 121-128, 254-262. (17) Huglin, M. B.; Rego, J. M. Macromolecules 1991, 24, 2556-2563. (18) Schulz, D. N.; Peiffer, D. G.; Agarwal, P. K.; Larabee, J.; Kaladas, J. J.; Soni, L.; Handwerker, B.; Garner, R. T. Polymer 1986, 27, 1734-1742. (19) Viklund, C.; Irgum, K. Macromolecules 2000, 33, 2539-2544. (20) Peters, E. C.; Sˇ vec, F.; Fre´chet, J. M. J. Adv. Mater. 1999, 11, 1169-1181.
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6; C7752); lysozyme, from chicken egg white (12650-88-3; L6876); and myoglobin, from horse heart (100684-32-0; M1882). All purchased proteins were stored according to the manufacturer’s recommendations and used as received. The antimicrobial peptide (enterocin A) separated in Figure 8 was obtained by cation exchange and hydrophobic interaction separation of a crude extract from Enterococcus faecium. The resulting sample, in 70% aqueous ethanol, was injected on the zwitterionic separation material. The biological activity of fractions collected that corresponded to each peak in the chromatogram was monitored by a bacteriocin-induction assay, as described by Aymerich et al.21 Photopolymerization. The zwitterionic monoliths were prepared by in situ photopolymerization using a procedure published elsewhere,19 with SPE as the zwitterionic monomer and TEGDMA or EDMA as the cross-linkers, methanol as the porogen, and benzoin methyl ether as the photoinitiator. The monoliths were prepared in DURAN glass columns (150 or 250 mm long × 2.62.7 mm i.d.; Kebo Lab, Stockholm, Sweden) and were anchored to the inner surface through graft polymerization onto methacrylate groups that had been introduced by reaction with neat 3-methacryloyloxypropyl trimethoxysilane. After completion of the polymerization, each column was furnished with fittings, connected to a LC system, and soluble compounds still remaining in the monolith were washed out using water as the mobile phase. Chromatography. Chromatography of proteins was accomplished by gradient HPLC using either a LDC (Laboratory Data Control, Riviera Beach, FL) system consisting of two LDC Constametric pumps, an LDC Gradient Master controller, and an LDC Spectromonitor 3100 variable-wavelength UV detector or a Bischoff (Leonberg, Germany) system comprising two HPLC compact pumps, a central processor, and a Lambda 1010 UV detector. The detectors were operated at 280 nm wavelength in both systems. Samples were injected through a Rheodyne (Cotati, CA) model 7125 loop injector, and data were collected using a Hewlett-Packard (Palo Alto, CA) HP3396A integrator. Flow rates and injection volumes were as indicated in each Figure. Separation of model proteins was carried out using gradient elution profiles as indicated in each figure legend. Model separations of inorganic ions were performed isocratically using an LKB 2150 pump (Amersham Pharmacia Biotech, Uppsala, Sweden) followed by an LDC ConductoMonitor for electrolytic conductivity detection using deionized water as the mobile phase. The effects of concentration, pH, and presence of chaotropic anions in the eluent were studied for three model proteins. Aliquots (20 µL) of lysozyme, cytochrome c, and R-chymotrypsinogen A in distilled water were separately injected at mobile phase compositions indicated in Figures 4 and 7 using a flow rate of 0.5 mL/min and isocratic conditions. The sodium perchlorate solutions were unbuffered to avoid eluent contributions from the buffer salt, but were adjusted to correct pH with NaOH or HNO3. The pH of these eluents after passage through the column was measured to ascertain that the deviations from the desired pH were less than 0.1 pH unit. Protein solutions for injection were prepared on a daily basis in water and stored in a refrigerator. Generally, triplicate injections were made of each protein at each mobile (21) Aymerich, T.; Holo, H.; Håvarstein, L. S.; Hugas, M.; Garriga, M.; Nes, I. F. Appl. Environ. Microbiol. 1996, 62, 1676- 1682.
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phase composition, and the capacity factor, k′, was calculated as:
k′ ) (tr,mean - tm)/tm Peptide purification chromatography was carried out with a linear gradient eluent ranging from pure water to 10 mM sodium phosphate buffer, pH 7.0. These experiments were performed using a Spectra-Physics (Mountain View, CA) SpectraSYSTEM P400 equipped with a Spectrafocus forward optical scanning detector. Scanning Electron Microscopy. Prior to the scanning electron microscopy study, the polymer samples were placed on sticky carbon foils which were attached to standard aluminum specimen stubs. The samples were coated with approximately 20 nm of gold by using a combination of sputter coating (Edwards S150A sputter coating unit, Edwards high vacuum, incorporating an automatic tilting and rotation device). Microscopic analysis of all samples was carried out in a S-360 iXP SEM (Leo Electron Microscopy Ltd., Cambridge, U.K.) operated in LaB6-mode, 10 kV, 100 pA probe current, and 0° tilt angle. RESULTS AND DISCUSSION Preparation of Zwitterionic Monoliths by Direct Copolymerization. N,N-Dimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPE) is a crystalline monomer that dissolves readily in water, although it is insoluble in many crosslinking monomers and most organic solvents commonly used as porogens in suspension or mold polymerizations.20 As we have described elsewhere,19 methanol can be preferably used because it dissolves all nonpolymeric components included in the mold as well as effectuating precipitation of a polymeric structure with suitable porosity. High concentrations of dimethacrylate crosslinkers and sulfoalkylbetaine monomer could, thus, be used without compatibility problems or precipitation of nonpolymer components. Photopolymerization was recently introduced by our group as a way of preparing monolithic materials22,23 and is a versatile route for the fast preparation of shallow layers of porous monolithic structures in columns or other confinements that are translucent to the 360-nm UV light that is used for initiating the polymerization. SEM micrographs of a representative copolymerized monolith show a monolith structure with macropore channels intersecting polymeric clusters comprised of coalesced spheroid units whose diameters range from 1 to 3 µm (Figure 1). Loss of sulfur due to scission of the pendant sulfoalkylbetaine groups would be detrimental to the stoichiometric charge balance that is required for a true zwitterionic material and would yield a stationary phase with a net ion exchange capacity, which would be more similar to the mixed-mode or pseudo-zwitterionic stationary phases described in the literature.6,7 Elementary analyses on the materials used in this study have shown that this procedure yields monoliths with sulfur:nitrogen ratios close to the expected 1:1 ratio, which indicates the presence of a zwitterionic stoichiometry.19 Interaction Between SPE-Containing Monoliths and Inorganic Ions. Several workers have tried to explain the retention (22) Viklund, C.; Ponte´n, E.; Glad, B.; Irgum, K. Sˇ vec, F.; Ho ¨rstedt, P. Chem. Mater. 1997, 9, 463-471 (23) Ponte´n, E.; Viklund, C.; Irgum, K.; Bogen, S. T.; Nilsson-Lindgren, Å. Anal. Chem. 1996, 68, 4389-4396.
Figure 2. Separation of a 5-µL aliquot containing 2 mM of each of KCl, KSCN, and Ca(SCN)2, using pure water at a flow rate of 0.5 mL/min as eluent, electrolytic conductivity detection, and a column (150 × 2.6 mm i.d.) synthesized as described in the legend of Figure 1, with the exception that the monomer phase consisted of 33% SPE and 67% EDMA.
Figure 1. Scanning electron micrographs at two different magnifications (see bars in respective micrograph) of a zwitterionic monolith prepared from SPE and EDMA. Polymerization conditions: 3 parts of monomer containing 47% SPE and 53% EDMA, respectively; 7 parts of methanol and 1% benzoin methyl ether (with respect to the weight of the monomers); photopolymerization in a 2.6-mm i.d. glass tube for 1 h using 360 nm light.
mechanism that causes ionic species to elute in an ion-pair-like fashion from ODS columns that have been dynamically coated with zwitterionic detergents, as pioneered by Hu.13 Such hydrophobic silica-based sorbents, noncovalently coated with detergents having a hydrophobic tail and a hydrophilic zwitterionic head, differ fundamentally from the presently discussed monolithic columns in which the backbone is a comparatively hydrophilic methacrylate polymer with a high fraction of the monomers copolymerized into the resin carrying covalently bonded sulfoalkylbetaine moieties. Our group has recently shown that sulfoalkylbetaine zwitterionic functionality can be introduced covalently onto 2-hydroxyethyl methacrylate beads using a two-step reaction involving activation with epichlorohydrin, followed by reaction of the epoxide group with 2-dimethylaminoethane sulfonic acid inner salt.14 Using this concept, the cation and anion components of inorganic salts could be separated individually or simultaneously when low concentrations of sodium perchlorate or perchloric acid were used as the eluent. However, this surface-functionalized material did not produce separation of inorganic anions using pure water as the mobile phase, a separation that can be achieved with ODS columns coated by zwitterionic detergents, as described by
Hu et al.13 Taking the disparity between the results of the above studies into consideration, it was essential to monitor the retention behavior of inorganic ions on the SPE based monoliths, even if the main target of the currently presented materials is in the field of bioseparations. Using pure water as mobile phase, it can be concluded that sulfobetaine-based monoliths retain monovalent cations less strongly than divalent and trivalent cations, respectively (data not shown). A mixture containing 2 mM each of KCl, KSCN, and Ca(SCN)2 was then injected and the resulting elution profile is shown in Figure 2. Injection of KCl and KSCN gave a chromatogram containing two peaks, that is, the salts were separated with respect to the difference in the anion, whereas a mixture of KSCN and Ca(SCN)2 produced two peaks that corresponded to the difference in cation identity. Overall, the elution order corresponded to that reported for silicas that are dynamically modified with sulfoalkylbetaine detergents.13 When 2 mM sodium perchlorate was used as the mobile phase, a salt mixture containing 1 mM each of KCl, KI, and KSCN gave a chromatogram that consisted of peaks that correspond to Cl-, I-, and SCN-, respectively, as well as a dip below the baseline that corresponds to the system peak (not shown). This is analogous to the HEMA material that was modified to carry covalently bonded sulfoalkylbetaine zwitterionic groups presented by our group.14 We, thus, conclude that the copolymerized sulfobetaine monoliths behave like strong/strong zwitterionic sorbents. Spatial Distribution of Zwitterionic Groups in Copolymerized Monoliths. In a mold copolymerization, it is difficult to determine whether functional groups are mostly buried in the matrix or efficiently exposed on the pore surface. Large molecules such as proteins may alter their conformation and local charge density in the vicinity of charged surfaces, and we, therefore, chose to probe the extent of accessible functionality by evaluating the retention for inorganic anions as a function of the SPE Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
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Table 1. Retention of Inorganic Ions on Copolymerized Zwitterionic Monoliths. SPE contentsa
capacity factorb, k′
% w/w
NaSCN
Ba(SCN)2
13 23 33 43
0.26 0.45 0.68 0.78
0.45 1.35 1.97 2.24
a Percentage of SPE monomer with respect to the weight of the monomers in the original polymerization mixture,which consisted of 30% monomer mixed with 70% methanol and 1% benzoin methyl ether. b Dependency of capacity factors for inorganic ions on the SPE concentration used during the polymerization of sulfoalkylbetaine-based monoliths. Five microliters of a solution containing 5 mM BaCl2 and 10 mM NaSCN was injected into the eluent, which was pure water pumped at a flow rate of 0.5 mL/min. Ions were detected in the effluent by electrolytic conductivity detection.
concentration in the polymerization mixture. The capacity factors for NaSCN and Ba(SCN)2 are shown in Table 1 using pure water as the mobile phase, where the expected increase in capacity factor with increasing admixture of SPE in the polymerization mixture is evident. Over the range tested in these experiments, the retentive properties of the SPE-based monoliths can, thus, be altered simply by varying the ratio between SPE and the crosslinker. The retention should also provide an indication of the density of functional groups that are accessible to solutes in aqueous solutions. Control of Protein Retention by Chaotropic Anions. Our main target when developing this new type of stationary phase was to develop a class of polymers with porous properties and surface chemistries for bioseparations using mild chromatographic conditions. The fundamental aspects of the interactions between proteins and sulfoalkylbetaine type monolithic polymers were recently shown in a study that was focused on the physical properties of monolithic sulfobetaine polymers.19 The interaction mechanism was found to be principally electrostatic in nature, as deduced from decreased interaction strengths for basic model proteins (R-chymotrypsinogen A, cytochrome c, and lysozyme) when the solution-phase salt concentration was increased. We also reported that the presence of chaotropic ions (perchlorate and thiocyanate were tested) have a complex influence on the properties of the sulfobetaine polymers. For example, an increased protein uptake capacity was monitored for poly(SPE-co-EDMA) monoliths when sodium perchlorate was added to the solution phase, and the permeability of poly(TRIM) monoliths carrying sulfobetaine groups attached in a tentacle arrangement was reversibly decreased as a consequence of adding even moderately chaotropic ions. Also, the interactions between the polymer and basic proteins were enhanced by addition of chaotropic ions to the solution phase. In this paper, we thus want to explore how these basic findings can be applied to protein separation chromatography. Owing to the net zero charge of strong/strong zwitterionic groups, it is reasonable to assume that the interactive forces between poly(SPE)-based monoliths and proteins were relatively weak. A separation of four model proteins using a gradient ranging from pure water to 2.5 mM phosphate buffer (pH 7) on a 250 × 2.6 mm i.d. poly(SPE-co-EDMA) column is shown in Figure 3. 448 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
Figure 3. Separation of a protein mixture containing 0.5 mg/mL each of myoglobin, R-chymotrypsinogen A, cytochrome c, and lysozyme. Conditions: 5-µL loop volume; 1 mL/min flow rate; linear gradient from pure water to 2.5 mM phosphate buffer, pH 7, in 9 min; UV detection at 280 nm. Column (150 × 2.6 mm i.d.) synthesized as described in the legend of Figure 1.
Noteworthy is the extremely low ionic strength required to elute even the most strongly retained protein (lysozyme) from the column. Further, it should be noted that when pure water was used as eluent, we did not see basic proteins elute, regardless of chromatographic run time, as opposed to inorganic ions which were eluted with water only as eluent. This is expected because the autoprotolytic process leading to self-elution would be considerably less facile for ions having multiple charge interactions with the stationary phase. Salt strengths this low will undoubtedly be advantageous for many separation applications. In other situations, such as in the separation of biologically active proteins that require a certain ionic strength or buffer type to preserve structural integrity, an eluent consisting of almost pure water might introduce problems. This restriction can be conveniently circumvented by utilizing the increased interaction strength between proteins and surfaces with sulfoalkylbetaine pendant groups that develops when chaotropic ions are added to the solution phase.19 Thus, chaotropic ions can be added to the eluent as a means of controlling the retentive power of the stationary phase, as demonstrated in Figure 4. The concentration of phosphate buffer (pH 7) required to elute lysozyme and cytochrome c from a poly(SPE-co-EDMA) monolith was considerably higher in eluents in which 10 mM sodium perchlorate or 10 mM sodium thiocyanate had been added, as compared to eluents without chaotropic ions. Perchlorate had the strongest influence on the protein retention, but thiocyanate was also effective in controlling the retentive power of the sulfobetaine monolith. The effect was quite substantial; the concentration of phosphate buffer needed to elute lysozyme increased by a factor of 10 when perchlorate was added. Evident from the Figure is also an increased resolution between the proteins in the presence of chaotropic ions, which provides a means for optimizing the chromatographic resolution of complex mixtures.
Figure 4. Dependence of protein (1.0 mg/mL injected separately through a 20-µL loop) retention on the concentration of phosphate buffer in the mobile phase as determined for lysozyme (closed symbols) and cytochrome c (open symbols). Phosphate buffer (pH 7), without addition of chaotropic eluent modifier ions (b/O), and with the addition of 10 mM sodium perchlorate (9/0) or 10 mM sodium thiocyanate (2/4). The poly(SPE-co-EDMA) monolith (150 × 2.7 mm i.d.) was synthesized using the composition described in the legend of Figure 1.
The results just discussed were obtained in isocratic runs with one protein injected at a time, while biopolymer separations generally are carried out in gradient mode. A phosphate buffer gradient with concentration ranging from 6 to 135 mM in 15 min with 10 mM sodium perchlorate added yielded an excellent separation of three model proteins on a poly(SPE-co-EDMA) monolith. A similar gradient without perchlorate addition resulted in elution of all of the proteins in the void volume, despite the overall lower ionic strength of the buffer (Figure 5). These findings, thus, provide us with possibilities of adjusting the strength and composition of the eluent to match different requirements with respect to protein nativity, protein resolution optimization, and detection considerations. Still, the proteins can be eluted by low-ionic-strength solutions within the entire experimental domain, which might be advantageous in preparative mode applications in which the biological activity should be preserved, as well as in salt-sensitive detection systems such as LC/MS. The promotion of retention by chaotropic ions suggests another way of controlling retention in a gradient elution, namely, by decreasing the chaotropic ion concentration while keeping a constant low buffer concentration. In order for this to work, the chaotropic ions attached to the zwitterionic stationary phase would have to be depleted through self-association of adjacent zwitterions or by autoprotolysis of water according to Scheme 1, processes that could be slow. However, in view of the forward and reverse breakthrough curves that we presented in a recent work for perchlorate ion on a polymeric sulfoalkylbetaine zwitterionic separation material for small ion separation,14 adsorption and release of perchloric acid is still a fairly rapid process. We, therefore, ran gradients of decreasing sodium perchlorate con-
Figure 5. Separation of a protein mixture containing 1.0 mg/mL each of myoglobin, cytochrome c, and lysozyme. Conditions: 20 µL loop volume, eluent at 0.6 mL/min flow rate; (a) linear gradient from 0 to 100% of eluent B (135 mM phosphate buffer, pH 7) in 15 min using a constant concentration of 10 mM sodium perchlorate in the eluent; (b) as in (a), but without addition of perchlorate ion. UV detection at 280 nm. Column as described in the legend of Figure 3.
centration at varying gradient times, resulting in the chromatograms that are presented in Figure 6. It is apparent that when rapid gradients were used (2 and 5 min), the proteins were eluted well after the programmed gradient had reached zero concentration of sodium perchlorate. Yet, the proteins are separated in both cases, and the separations seem to be nearly identical. With a considerably slower gradient (30 min; data not shown), the elution of cytochrome c took place after 14.8 min, that is, while the chaotropic eluent component was still being pumped. All of these data indicate that the proteins are retained as long as a sufficient amount of perchlorate ion is associated with the zwitterionic separation material and that the order of elution coincides with the binding strength as a function of the perchlorate ion concentration, as discussed above. The chaotropic effect of acid counteranions added to the eluent in reversed-phase chromatography of basic peptides has been reported by Sereda et al.24 They explained the retention increase by the increased hydrophobicity that is obtained when perchlorate ions interact with basic proteinaceous solutes, causing a disturbance of the environment surrounding the solvated analyte. Interaction between basic analytes and perchlorate anions can, thus, be a result of direct electrostatic interaction (ion pairing), as well as water structure breaking effects. It would be reasonable to assume that the retention increase brought about by chaotropic anions on the polymeric stationary phase described in this work could be driven by solvophobic effects, as well as electrostatics. However, this assumption does not hold experimentally because (a) the retention increase is seen also for proteins exhibiting very low retention in hydrophobic interaction chromatography (HIC); (b) upon addition of chaotropic anion, the interaction strength reaches a plateau, after which further addition causes the interaction strength between the protein and the stationary phase to (24) Sereda, T. J.; Mant, C. T.; Hodges, R. S. J. Chromatogr. A 1997, 776, 153165.
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Scheme 1. Schematically Depicted Elution Processes Mediated by (A) Water Splitting, and (B) Zwitterion Self-Association.
Figure 6. Separation of cytochrome c and lysozyme using a decreasing gradient of sodium perchlorate at two different gradient rates. Conditions: 20-µL loop volume; 0.6 mL/min flow rate; linear gradients from eluent A (10 mM sodium perchlorate in 5 mM phosphate buffer, pH 7) to eluent B (5 mM phosphate buffer, pH 7) in 2 or 5 min, respectively, as shown in the Figure. UV detection at 280 nm. Column as described in Figure 3.
decrease with ionic strength analogous to conventional ion exchange;19 (c) high protein recoveries indicate low losses due to resin hydrophobicity (see below); and (d) ion pairing with perchlorate ions should render the cationic proteins more neutral and, thus, less prone to ionic interactions. Instead we ascribe an explanation model based on the antipolyelectrolytic behavior of linear sulfobetaine polymers, which translates into an increase in solubility and viscosity with increasing salt concentration. This electrolyte dependency follows the Hofmeister lyotropic series for anions’ capability of salting in macromolecules from aqueous solutions15,16,19. We, thus, suggest that the retention of basic proteins on the poly(SPE-co-EDMA) monoliths was increased due to an increased ion exchange capacity of the sulfobetaine units in the presence of chaotropic anions. This interpretation is supported by experiments with small ions on sulfoalkylbetaine zwitterionic separation materials in which the apparent cation exchange capacity was strongly affected by millimolar concentrations of perchloric acid.14 The data from the reverse gradient in Figure 6 also support this interpretation. The effect of eluent pH was examined for the model proteins using aqueous sodium perchlorate mobile phases where the pH 450 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
was varied between 4.5 and 10.2 through addition of sodium hydroxide or nitric acid. The results of these experiments are shown in Figure 7 and demonstrate that the retentions of R-chymotrypsinogen A, cytochrome c, and lysozyme were pHdependent and that the capacity factors for these three proteins increased as the pH reached lower values. This indicates that these basic proteins interact with the zwitterionic phase in a mode closely resembling cation exchange. Application of Sulfobetaine Monoliths in Peptide Purification. Separation of natural or synthetic peptides is of tremendous importance in various aspects of biomedical science. The goal of the separation may be isolation, purification, identification, or purity determination of the peptide.25 The applicability of media with sulfoalkylbetaine zwitterions as novel interaction layers for peptide purification and analysis was tested for a bacteriocin, which had been partially purified from E. faecium according to established methods26 (omitting the final reversed-phase separation). These peptides are hydrophobic and basic in nature, and the (25) Irgum, K. In Meyers, R. A., Ed.; Encyclopedia of Analytical Chemistry; Wiley: Chichester, U.K.; in press. (26) Casaus, P.; Nilsen, T.; Cintas, L. M.; Nes, I. F.; Hernandez, P. E.; Holo, H. Microbiology 1997, 143, 2287-2294.
Figure 8. Purification of 100 µL protein extract containing an unknown amount of biologically active antibacterial peptide in 70% ethanol. Biological activity was detected in the two fractions collected around the peaks marked by arrows. Conditions: linear gradient from pure water to 10 mM phosphate buffer, pH 7, in 10 min at a flow rate of 0.7 mL/min. Detection was carried out by UV absorption spectrometry at 214 nm. Column (250 × 2.7 mm i.d.) prepared as described in the legend of Figure 1.
fractions eluting from the column. The chromatogram was recorded by monitoring the absorbance at 280 nm, and it is obvious that a significant amount of nonactive proteins or peptides were eluted close to the void volume. Protein Recovery. Nonspecific interactions between proteins and stationary phases may cause severe tailing and peak distortion, as well as decrease the effective throughput in preparative mode. Lysozyme is a protein of moderate size with a hydrophobic region and is, therefore, suitable as a probe for residual hydrophobic interaction of separation media.27 For the SPE-co-EDMA monolith used in Figure 7, the recovery of the more hydrophilic protein cytochrome c was determined to be 96% for a 20-ng injection, while the recovery for the same amount of lysozyme was above 85% with the fully aqueous eluents used. Figure 7. Dependency of protein retention on the mobile-phase pH, as determined for lysozyme, R-chymotrypsinogen A, and cytochrome c (1.0 mg/mL, respectively) injected through a 20-µL loop using sodium perchlorate adjusted to various pH values ranging from 4.5 to 10.2. Column preparation as described in the legend of Figure 1.
existing purification schemes call for reversed-phase chromatography using up to 70% 2-propanol in the eluent. A chromatogram showing the purification of 100 µL extract containing the antibacterial peptide on a poly(SPE-co-EDMA) monolith is shown in Figure 8. Antibacterial activity was found in two fractions corresponding to the peaks eluting at 10.3 and 10.9 min in the chromatogram, as determined by agar plate assay monitoring of
CONCLUSIONS Porous monoliths carrying zwitterionic sulfoalkylbetaine groups can be prepared by in situ photopolymerization using SPE and a suitable cross-linking monomer. The resulting monolithic copolymer was capable of reversible ionic interactions with proteins and peptides in totally aqueous eluents. Owing to a favorably weak interaction strength and the absence of hydrophobic interactions, fast separations of basic proteins and peptides could be realized using exceptionally mild conditions. Of particular interest is the possibility of modulating the apparent net surface charge on the (27) Mu ¨ ller, W. J. Chromatogr. 1990, 510, 133-140.
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sulfoalkylbetaine polymer using small amounts of perchlorate or thiocyanate ions in the eluent. This property, thus, provides a unique way of controlling the polymer-protein interaction strength over a wide range on a single monolith and consequently widens the scope of the separation process. In an ongoing study, we are aiming toward further improvements of the selectivity of zwitterionic monoliths with respect to acidic proteins by incorporating different zwitterionic groups, and by utilizing a grafting route instead of direct copolymerization. ACKNOWLEDGMENT Per Ho¨rstedt is gratefully acknowledged for his valuable help with the scanning electron microscope analyses, as is Michael
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Sharp for linguistically revising the manuscript. Appreciation is also directed to the Bengt Lundquist Memorial Foundation (C.V.), the J.C. Kempe Memorial Foundation (C.V.), and the Magnus Bergwall Foundation (K.I.) for providing financial support. This work was supported by grant K-KU 8735-315 from The Swedish National Science Research Council.
Received for review May 31, 2000. Accepted November 1, 2000. AC000618R
