Anal. Chem. 1998, 70, 3348-3354
High-Performance Membrane Chromatography of Supercoiled Plasmid DNA Roberto Giovannini and Ruth Freitag*
Laboratoire de Biotechnologie Cellulaire, Ecole Polytechnique Fe´ de´ rale de Lausanne, 1015 Lausanne, Switzerland Tatiana B. Tennikova
Institute of Macromolecular Compounds, Russian Academy of Sciences, 199 004 St. Petersburg, Russia
Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the threes albeit isolatedsbands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 × 52 mm, 10-µm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.
High-performance membrane chromatography (HPMC) differs from conventional (column) chromatography in the geometry and the structure of the stationary phase. The typical stationary phase in HPMC is a flat disk (diameter in the centimeter range, thickness in the micro- to millimeter range) bearing a large number of throughpores with similar, optimized diameter.1 The technique was first suggested more than a decade ago. Its continuing optimization depends on the consequent application of our developing understanding of the specific nature of biopolymers to the theory of chromatography.2 For example, proteins tend to vary widely in their interaction energy with a stationary phase as documented inter alia by their (1) Tennikova, T. B.; Svec, F. J. Chromatogr. 1993, 646, 279. (2) Tennikova, T. B.; Freitag, R. In Analytical and Preparative Separation Methods for Biomolecules; Aboul-Enein, H. Y., Ed.; Marcel Dekker: New York, in press.
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Z-parameters in ion-exchange chromatography. As a consequence, gradient rather than isocratic elution chromatography must normally be employed for their separation. Because of that, the actual operative thickness of adsorptive layer (OTAL) is short in protein chromatography, much shorter than the length of a typical chromatographic column. Due to the size of the analytes, protein chromatography tends to be much more sensitive to masstransfer and steric effects than does chromatography of small molecules. In HPMC, the “column length” is in the range of the OTAL and from the many mass-transfer effects dominating conventional chromatography only the diffusional migration through the boundary layer of the liquid on the wall of the pores to the adsorptive surface remains important. HPMC thus emerges as the perfect tool for fast, high-resolution protein chromatography. If at all, high flow rates have a beneficial effect on the separation.3 Small molecules, on the other hand, are almost impossible to separate by HPMC, since this would require repetitive adsorption/ desorption events.4 Why this should be the case can only be understood by the rigorous application of the theory of HPMC. Such a theory has been developed only recently,2,5,6 and up to now the fact that the more “easy” separations (i.e., that of mixtures of small molecules) give poor results has prevented many potential beneficiaries of HPMC from using the method. The previous lack in a sound theoretical basis has certainly contributed to the existence of a most prominent “blank spot” in the area of biopolymer HPMC. While protein HPMC is rapidly evolving as an analytical and (semi)preparative technique, the separation of polynucleotides in any form or configuration is simply not an issue. While the overwhelming majority of plasmid preparations from bacterial lysates continue to rely on methods such as ultracentrifugation and extraction, chromatography in general is increasingly been discussed as a more efficient way to obtain pure DNA.7-11 However, in the area of DNA/plasmid(3) Tennikov, M. B.; Gazdina, N. V.; Tennikova, T. B.; Svec, F. J. Chromatogr., A 1998, 798, 55. (4) Svec, F.; Frechet, J. M. J. Macromol. Symp. 1996, 110, 203. (5) Tennikov, M. B. Chem. Phys., submitted for publication. (6) Tennikov, M. B.; Tennikova, T. B. Anal. Chem., submitted for publication. (7) Perbal, B. In A Practical Guide to Molecular Cloning; Wiley: New York, 1984; p 165. (8) Ohmiya, Y.; Kondo, Y.; Kondo, T. Anal. Biochem. 1990, 189, 126. (9) van Helden, P. D.; Hoal, E. G. In New Nucleic Acid Techniques; Walker, J. M., Ed.; Humana Press: Clifton, NJ, 1988; p 61. S0003-2700(98)00390-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 07/22/1998
HPMC only a single paper exists, which considers the plasmid preparation from a bacterial lysate.12 While being ingenious, this paper could equally well be considered a contribution to protein separation. The specifics of DNA (or RNA) molecules under HPMC conditions are neither investigated nor considered. In our opinion, such an investigation is necessary for the development of polynucleotide HPMC, especially since the results presented in this paper show a pronounced difference in the chromatographic behavior of DNA molecules, and most prominently so of plasmid DNA molecules, as compared to that of proteins. The objective of the investigation of the HPMC of plasmid DNA was manyfold. One aspect was the possibility of initiating the development of a new and powerful fast analytical/preparative method for the separation of this valuable material for biomedical (gene therapy) or biotechnological (transfection) applications. In addition, chromatography and especially HPMC also presents a unique possibility to study the dynamic behavior of these highly charged and densely structured supermolecules. The information obtained could be interesting from the point of view of the physical, chemical, and biological sciences. Last, a comparison of the chromatographic behavior of these simple biopolyelectrolytes with that of the more complex proteins will advance the general understanding of HPMC. EXPERIMENTAL SECTION Materials. High-purity water and chemicals of analytical grade quality were used throughout. Plasmids (pCMVβ, Clonetech, Palo Alto, CA) were produced in Escherichia coli (DH5R) grown in LB medium (Gibco Life Technologies Inc., Gaithersburg, MD) containing 50 µg/mL ampicillin. After alkaline lysis, predominantly supercoiled plasmids were purified by CsCl density gradient centrifugation (1×) according to standard laboratory practice. The plasmid sample used in the chromatographic experiments had 7.2 kb. The OD 260/280 was 1.8 ((0.03) for all plasmid preparations. Stationary phases (disks) for gradient and isocratic anionexchange HPMC were from BIA doo, Ljubljana, Slovenia. Disks had a diameter of 12 mm and a thickness of 3 mm. The base material is a macroporous polyglycidyl methacrylate-co-ethylene dimethacrylate) (GMA-EDMA) matrix. Both strong anionexchanger bearing quaternary ammonia groups (Q-AE type) and weak anion-exchanger disks bearing diethylaminoethyl groups (DEAE-AE type) were used. The former were purchased as such from BIA; the latter were prepared from epoxy disks also commercially available from BIA, by the following procedure. The epoxy disks were immersed in 2 mL of pure diethylamine in a small, covered bottle. The reaction was allowed to proceed at ambient temperature for 24 h without any stirring. Afterward, the disks were washed excessively with pure water. Prior to a chromatographic experiment, disks were equilibrated with the respective starting buffer. The functional groups density was 1.5 mmol/g for both disk types. One disk unit contains 0.18 g of polymer (density 1.06 g/mL). For comparison, a continuous-bed column (UNO Q1, dimensions 7 × 35 mm, functional group density 0.414 mM) and an (10) Moreau, N.; Tabary, X.; Le Goffic, F. Anal. Biochem. 1987, 166, 188. (11) Flanagan, J. M.; Fujimara, R. K.; Jakobson, K. B. Anal. Biochem. 1986, 153, 299. (12) van Huynh, N.; Motte, J. C.; Pilette, J. F.; Decleire, M.; Colson, C. Anal. Biochem. 1993, 211, 61.
analytical-scale column packed from porous 10-µm particles (BioScale Q2, dimensions 7 × 52 mm, functional groups density 0.217 mM) were used. The functional group densities were determined by salt breakthrough curves. Both strong ionexchanger-type columns bore quaternary ammonium groups (QAE) and were donated from BioRad, Hercules, CA. Instrumentation. The chromatographic system consisted of two piston pumps (Irica Σ 871, ERC, Alteglofsheim, Germany), a UV detector (S-3702, Soma Optics Ltd., Tokyo, Japan) with the wavelength set to 260 nm, a mixing chamber (ERC, Alteglofsheim, Germany), and a six-port valve (Valco, Houston, TX). The data were processed with a Chromatography Station for Windows software program (Data Apex Ltd., Techlab, Braunschweig, Germany). For the insertion of the disks into the chromatographic system, specially designed cartridges were purchased from BIA doo. The gel electrophoresis equipment was from Hoefer Scientific Instruments, San Francisco, CA. Methods. Unless indicated otherwise, procedures given in ref 13 were used for DNA preparation and analysis. The integrity of the DNA was confirmed by 0.8% agarose gel electrophoresis (10 cm, 5.8 V/cm). A 1-kb ladder (Gibco Life Technologies Inc.) was used as the molecular weight marker. Plasmids were linearized with ScaI (Gibco Life Technologies Inc.) and recovered by ethanol precipitation. Samples for chromatographic experiments were prepared to have plasmid DNA concentrations of 1 µg/µL in 10 mM Tris HCl buffer, pH 8.0, containing 1 mM EDTA (TE buffer). If indicated, the samples were further diluted with buffer A. For gradient elution experiments buffer A was a 20 mM Tris HCl buffer, pH 7.4, and buffer B a 20 mM Tris HCl buffer, pH 7.4, containing an additional 1 M of NaCl. For isocratic separations, defined mixture of buffers A and B served as mobile phase. A detection wavelength of 260 nm was used throughout. RESULTS AND DISCUSSION Plasmid DNA molecules are biopolymers and more specifically biopolyelectrolytes. This has important consequences for their solubility in water and for their isolation by chromatography. Just like proteins, plasmids diffuse slowly and show a pronounced steric effect. In contrast to proteins, they bear only negative charges, which are evenly distributed over the molecule. Thus, their chromatographic behavior should be easier to understand than that of proteins, where both positive and negative charges usually contribute to the overall net charge and where a surface characterized by an uneven distribution of charges and local charge clusters is the rule rather than the exception. Unlike many synthetic polyelectrolytes, which share with DNA the homogeneous charge structure, all plasmid molecules of a given type are supposed to be identical in their molecular mass. Gradient Elution Experiments. Protein disk chromatography is only possible in the gradient elution mode. When similar conditions were used for plasmid DNA, chromatograms such as the one depicted in Figure 1 ensued. The plasmid interacts strongly with the stationary phase and desorbs only when a high percentage of buffer B (high salt concentration) is reached. (13) Sambrook, I.; Fritsch, E. F.; Maniatis, T. Molecular cloning, a laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.
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Figure 1. Gradient elution HPMC of plasmid DNA. Conditions: stationary phase, Q AE disk; flow rate, 1 mL/min; gradient, 0-5 min 100% buffer A, 5-10 min 100% buffer A to 100% buffer B 12-13 min 100% buffer B, 13-18 min 100% buffer B to 100% buffer A; sample, 10 µL (corresponding to 2 µg of plasmid DNA (pCMVβ)). Inset: agarose gel electrophoresis. Line 1 corresponds to the DNA ladder; lines 2 and 3 correspond to two samples of plasmid pCMVβ.
The current model of HPMC interprets the separation as a single-step desorption process.2 The resulting peak should closely correspond to the theoretical curve given by the probability of the passage of a desorbing molecule from the stationary to the mobile phase in a certain mobile-phase modifier concentration. Since the peak in Figure 1 is sharp, we can assume a narrow distribution of the adsorption energies among the molecules of the plasmid preparation. This is to be expected given the simple chemistry of DNA. However, the peak shows a shoulder, so the presence of at least two fractions with different adsorptive abilities in the sample can be speculated upon. From the agarose gel electrophoresis, we deduced that the supercoiled plasmid preparation contained two impurities, presumably nicked and open circular DNA, Figure 1 inset. Some residual genomic DNA also seemed to be present. It is likely that some of these conformations differed enough from the supercoiled one in adsorption energy to show in the chromatogram. A first investigation of the concentration effect gave an excellent linear correlation between the peak height and the injected amount between 2 and 10 µg (y ) 669.5x + 4.28, r ) 0.9988). Sample volumes were 10 µL in all cases; the gradient conditions were as given in Figure 1. A 10-µg sample was still well within the linear range of the calibration curve. Concentrations below 2 µg were not investigated since the LOD was rapidly approached. The linearity of the calibration curve indicates that the desorption process is complete (100% recovery) and that no nonspecific interaction between the DNA and polymer surface occurs. At the same time, it was verified that the mobile-phase flow rate could be raised from 1 mL/min to at least 4 mL/min without adverse effect on the efficiency (data not shown). This flow rate independence of the separation efficiency is one of the distinct advantages HPMC has in protein chromatography. Since little diffusion takes place, the most prominent effect of an increase in the flow rate, as was recently shown both experimentally and theoretically, is an increase in the active pore surface, since more pores act as flow-through pores, when a higher pressure is applied to enforce a more rapid flow rate.3 3350 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 2. Influence of the starting buffer composition in gradient elution of plasmid DNA. Conditions: stationary phase, DEAE AE disk; flow rate, 4 mL/min; gradient, linear from 77 to 100% B (peak 1), 75 to 100% B (peak 2), and 73 to 100% B (peak 3); gradient time, 2 min; sample, 10 µL (corresponding to 5 µg of plasmid DNA (pCMVβ)).
Due to the presence of only negative charges, DNA molecules are usually adsorbed more strongly on anion-exchange stationary phases than proteins. In protein HPMC, an adsorption buffer of low ionic strength (buffer A) is usually prerequisite to achieve retention during loading. In contrast, the strong interaction of the plasmid DNA allowed experiments directed toward a decrease in the binding strength. Both a change in composition of the adsorption buffer from pure buffer A to mixtures of buffers A and B and the use of different interactive groups of the stationary phase (weak DEAE groups instead of strong Q groups at the same surface density) were investigated. The effect of such a fine-tuning of the DNA surface interaction during adsorption is shown in Figure 2. Adsorption on the DEAE disks is still possible even if a starting buffer containing up to 77% buffer B and only 23% buffer A is used. Interestingly, the elution pattern changes considerably between 23 and 27% buffer A. Depending on the exact conditions, we observed a peak with three maximums (trace 3 in Figure 2), or two maximums and a more or less pronounced shoulder (traces 1 and 2 in Figure 2), where in Figure 1 we had seen a major peak with a shoulder for the same sample. The presence of three maximums corresponds well to the presence of three fractions in the agarose gel of the plasmid (Figure 1 insert), which were assumed to stem from the supercoiled (major band), nicked, and open circular fraction. However, the chromatographic separation was far from satisfactory and did not allow separation of the three types of DNA. At present electrophoresis is still required for that. When the gradient shape and composition was kept constant (73-100% B) but the gradient slope was decreased at a constant flow rate of 3 mL/min, yielding gradient volumes from 6 to 43.5 mL, the resulting chromatograms, shown in Figure 3, became even more complex. In all cases, the three major fractions were observed at constant ratios. However, as we proceeded to larger gradient volumes, an additional maximum appeared in a reproducible manner. It is known that the supercoiled conformation can exist in several variations of the tensed molecular structure. It is possible that the additional peak in the chromatogram for larger gradient volume was caused by these forms.14 (14) Oana, H.; Hammond, R. W.; Schwinefus, J. J.; Wang, S.-C.; Doi, M.; Morris, M. D. Anal. Chem. 1998, 70, 574.
Figure 3. Gradient elution of plasmid DNA using different gradient slopes. Conditions: stationary phase, DEAE AE disk; flow rate, 3 mL/ min; gradient, linear from 73 to 100% B; gradient time, 2 (peak 1), 2.5 (peak 2), 3 (peak 3) 4.5 (peak 4), and 14.5 min (peak 5); sample, 10 µL (corresponding to 5 µg of plasmid DNA (pCMVβ)).
As the gradient became even shallower (trace 5 in Figure 3, gradient time 14.5 min, gradient volume 43.5 mL), another phenomenon was observed. The chromatogram became less “smooth” and minor peaks or spikes appeared, but this time in an nonreproducible, statistical fashion. This has also been observed in protein HPMC for very shallow gradients and in this case was explained by the assumption of an inhomogeneity of the adsorptive surface and small momentary energetic distribution within the protein molecules at the moment of adsorption.15 However, inhomogeneities in the mobile-phase composition could also give rise to the observed effect. The final answer to the problem will have to await further research. The flow rate has a similar influence on the chromatogram. When the gradient time was kept constant and the flow rate was decreased from 4 to 3 and 2 mL/min, the highest flow rate gave the smoothest chromatogram (data not shown). As was pointed out above, the appearance of three maximums in the plasmid chromatogram agrees well with a similar number of bands in the agarose gel of the same plasmid. If we assume the three maximums to be due to three conformations (supercoiled, nicked, open circular) of the same plasmid, a linearization of the molecules in the preparation should result in a homogenization of the mobilities and interaction energies, respectively. Figure 4 demonstrates that this is indeed the case. In comparison with the original peak (trace 2), the signal of the linearized plasmid (trace 1) is more homogeneous. Interestingly, it is also shifted toward shorter retention times; i.e., the interaction energy decreases as a consequence of the linearization. One explanation for this behavior might be that the “charge density” of the flexible linearized molecule is lower, which would result in a weaker interactive force. A similar change in adsorption strength can be observed for native and denatured protein molecules of otherwise identical primary structure. Another explanation takes into account the specific conditions under which interaction in HPMC occurs. In contrast to conventional chromatography based on molecular diffusion, shear effects cannot be disregarded in HPMC, where adsorption takes place on the “walls” of the pores through which the mobile phase is pumped. As a large molecule, DNA (15) Dubinina, N. I.; Kurenbin, O. I.; Tennikova, T. B. J. Chromatogr. 1996, 753, 217.
Figure 4. Comparison of linearized and native plasmid DNA in gradient elution. Conditions: stationary phase, DEAE AE disk; flow rate, 3 mL/min; gradient, linear from 73 to 100% buffer B in 4.5 min; sample, 10 µL (corresponding to 5 µg of linearized (peak 1) and nonlinearized (peak 2) plasmid DNA (pCMVβ)).
can be assumed to reach into the flow region. Differences in flexibility may influence the interaction under these conditions. Isocratic Elution HPMC. The pronounced difference between HPM chromatography of the two biopolymer classes, proteins and (plasmid) DNA, became obvious during the isocratic elution experiments. Isocratic separation of proteins is considered impossible in HPMC, since it is just the single-step desorption process achieved in a rapid gradient, which is responsible for their separation. Moreover, the fact that isocratic separations of molecules with similar interaction energies, which requires repeated adsorption/desorption events, are difficult or impossible is used as rational to explain the difficulty in separation of small molecules in HPMC. Nevertheless, we find that the isocratic separation of the plasmid conformations is easily achieved using the Q disks. Figure 5 combines a series of experiments, in which different eluents and flow rates were used. Obviously an eluent composed of 75% buffer B and 25% buffer A gives the best “separation”, although three maximums are also observed for eluents containing as much as 80% buffer B. Concomitantly, the elution occurs earlier at a given flow rate, when a higher percentage of buffer B is used. While the finding that isocratic separation of plasmid DNA was possible at all was already surprising, the observed effect of the flow rate was even more so. We have learned from protein separations that an increase in the flow rate should have either a beneficial or no effect on the separation.16-18 In contrast, a flow rate of only 1 mL/min seems to be the optimum in terms of speed and resolution in plasmid chromatography (Figure 5a). The use of higher flow rates, i.e., 2 and 3 mL/min (Figure 5b and c, respectively) led to a loss in resolution. The application of even smaller flow rates, such as 0.2 mL/min (Figure 5d), further reduced the smoothness of the chromatographic trace without improving the overall separation. To a certain extent these phenomena may be due to the fluid dynamics in HPMC. A very simple description of the influence of flow rate on HPMC separation can be based on a comparison of the residence time of the mobile phase (and the analytes) in (16) Strancar, A.; Koselj, P.; Schwinn, H.; Josic, D. Anal. Chem. 1996, 68, 3483. (17) Strancar, A.; Barut, M.; Podgornik, A.; Koselj, P.; Schwinn, H.; Raspor, P.; Josic, D. J. Chromatogr. 1997, 760, 117. (18) Reif, O.-W.; Freitag, R. J. Chromatogr. 1993, 654, 29.
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Figure 5. Influence of the buffer composition and the flow rate on isocratic plasmid DNA elution. Conditions: stationary phase, Q AE disks; sample, 10 µL (corresponding to 5 µg of plasmid DNA (pCMVβ)). (a) Flow rate, 1 mL/min. Peaks: (1) 100, (2) 95, (3) 90, (4) 85, (5) 80, (6) 78, and (7) 75% buffer B. (b) Flow rate, 2 mL/min. Peaks: (1) 100, (2) 95, (3) 90, (4) 85, (5) 80, and (6) 75% buffer B. (c) Flow rate, 3 mL/min. Peaks: (1) 100, (2) 95, (3) 90, (4) 85, (5) 80, and (6) 75% buffer B. (d) Flow rate, 0.2 mL/min. Peaks: (1) 100, (2) 85, and (3) 75% buffer B.
the disks and the time required for mass transfer of the analytes through the stagnant fluid layer to the adsorptive surface. Obviously the residence time must be long enough to allow diffusion of the analyte to the pore wall. Plasmid DNA molecules are rather large molecules and their diffusivity is low. The observed disappearance of the complex peak pattern at 3 mL/ min may result from the decrease in the residence time of the molecules within the disk, which no longer allows adsorption to occur. On the other hand, the dynamic sorption/desorption process is close to equilibrium as the DNA molecules move through the disk for flow rates such as 1 mL/min. Under these conditions, the residence time is sufficiently long for the DNA molecules to come into contact with the charged ligands on the pore walls. The pressure drop required to achieve a specific flow rate decreases with the square of the pore diameter. Although the disks used in the investigations are optimized for chromatography and have a very homogeneous pore structure and a relatively narrow pore size distribution, a pore size distribution still exists. Therefore the percentage of “active” pores increases with the flow. According to the Hagen-Poisseuille law, the flow velocity at a given pressure drop is proportional to the square of the pore radius. The distribution in pore size thus leads to a distribution in flow velocities within the different pores and to a concomitant peak broadening. Since at higher flow rates a larger number of pores is “flow-active”, the band-broadening effect is more pronounced. This can also be observed in the separation of the plasmid DNA, where the peaks at a flow rate of 2 and 3 mL/min are broader than those obtained at 1 mL/min. This effect differs 3352 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
substantially from the band broadening observed at higher flow rates in conventional chromatographic columns, where it is due to an increase in mass transfer resistance. Anion-Exchange Chromatography of Plasmid DNA in Standard Column Format. For the sake of comparison, we also used standard columns containing strong anion exchangers (QAE type). Specifically, we were in the position to include two types of column, a BioScale Q2 and the new UNO (Q1). The UNO column bears a certain similarity to our disks, since it is not packed with particles but according to the manufacturer consists of a monolithic porous rod. While the exact details of the composition of the UNO-type columns are proprietary, it is expected that these columns have a pore structure very similar to that of the chromatographic disks.19,20 Just as with the disks, we have a coincidence of flow pores and interactive surface. Therefore, the separation process in UNO columns can be similar in respect to the contribution, or rather the lack of it, of mass-transfer effects. However, the “separation layer thickness” is 35 mm in the case of the UNO column, compared to a mere 3 mm for the disks. Chromatography on a UNO column thus may be governed by multiple-step adsorption/desorption processes similar to mechanisms typical for standard column chromatography. The DNA fractionation under the same gradient elution conditions for the short separation layer of the disk and the UNO column are shown in Figure 6. It should be emphasized that the same linear flow velocities were used in both cases, corresponding (19) Kasper, C.; Meringova, L.; Freitag, R.; Tennikova, T. B. J. Chromatogr. 1998, 798, 65. (20) Vogt, S.; Freitag, R. Biotechnol. Prog., in press.
Figure 6. Comparison of plasmid fractionation using the Q AE disk and a continuous-bed column. Conditions: stationary phase, Q AE disk (1) and UNO Q1 column (2); flow rate, 1 (disk) and 0.5 mL/min (column); gradient, 0-5 min 100% buffer A, 5-10 min 100% buffer A to 100% buffer B, 12-13 min 100% buffer B, 13-18 min 100% buffer A; sample, 10 µL (corresponding to 2 µg of plasmid DNA, (pCMVβ)).
Figure 7. Isocratic elution of plasmid DNA using a continuous-bed column. Conditions: stationary phase, UNO Q1 column (7 × 35 mm); flow rate, 0.5 mL/min; eluent, 80% buffer A and 20% buffer B; sample, 10 µL (corresponding to 2 µg of plasmid DNA (pCMVβ)).
Figure 8. Comparison of plasmid DNA fractionation using disk, UNO column, and a packed-bed strong anion-exchange column. Conditions: stationary phase, BioScale Q2 column (7 × 52 mm, 10-µm porous particles), UNO Q1 column (7 × 35 mm), and Q-AE disk (12 × 3 mm); flow rate, 0.5 mL/min; sample, 10 µL (corresponding to 2 µg of plasmid DNA (pCMVβ)). (a) Gradient, 0-5 min 100% buffer A 5-10 min 100% buffer A to 100% buffer B, 10-12 min 100% buffer B, 12-13 min 100% buffer B to 100% buffer A, 13-18 min 100% buffer A; peak 1 BioScale Q2 column, peak 2 UNO Q1 column. (b) Stationary phase, BioScale Q2 column; eluent, 25% buffer B and 75% buffer A.
to a flow rate of 1 mL/min for the disks and of 0.5 mL/min for the column in order to achieve similar fluid dynamics in both systems. The desorption peak is sharper with the disk than with the column. This is surprising, since the multistep process typical for the column geometry is expected to lead to an averaging of the statistical variation in the interaction energy and thus to peak sharpening. In addition, the peak shape obtained with the column does not indicate the presence of any subfractions (shoulder) within the plasmid preparation. Gradient elution with varying compositions of the starting buffer similar to those that have been performed with the disks did not result in a fractionation of the plasmid sample on the UNO column either, except for a starting eluent consisting of 55% buffer A and 45% buffer B, for which a small shoulder on the back of the desorbing peak was observed. An isocratic elution, albeit using buffers with 25 and 20% buffer B, did result in a fractionation of the plasmid preparation similar to that found with the disks, Figure 7. Under isocratic conditions, the longer separation length of the rod column did improve the separation in terms of retention time differences of the respective maximums, however, at the price of a concomitant increase in zone broadening. The quantitative ratio of the three fractions is also slightly different from that observed for the disk. A discussion of these differences is difficult, because no sufficient informa-
tion concerning characteristics such as accessibility of the adsorptive surface or the pore size distribution for the UNO column is at present available. The picture changes again for the particle-based BioScale Q2 column. Under gradient conditions, an even broader peak than for the UNO column was observed, Figure 8a. This can be explained in terms that the plate height of the packed-bed column is more sensitive to chromatographic conditions such as the flow rate, especially when larger molecules are concerned. The difference between the monolithic stationary phases and the packed-bed column was even more pronounced in isocratic elution. Figure 8b does not reveal any fractionation of the plasmid preparation. This does not change even in eluents with varied composition. Peaks with a single maximum and no discernible shoulders were observed throughout. Again, the reason for this difference can only be speculated upon. However, the absolute numbers of interactive groups present in a given “column” seem to be comparable, i.e., 434 (BioScale), 538 (UNO), and 270 µM (disk), for all stationary phases considered. Unless a severe size exclusion effect for the BioScale column is responsiblesand we found no evidence for thissthe use of a monolithic block compared to a packed bed must be assume to influence chromatographic separations regardless of the column length. Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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ACKNOWLEDGMENT We thank Ms. Tiziana Flego and Ms. Alexandra Kulangara for the preparation of the plasmids. Professors Florian Wurm and William Chourchesne as well as Dr. Johanna van Adrichem are acknowledged for patient explanation of the specifics of plasmid DNA. Dr. Sabine Vogt determined the functional group densities
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of all column materials. The Ecole Polytechnique Fe´de´rale de Lausanne supported this work by a research grant to T.B.T. Received for review April 8, 1998. Accepted June 30, 1998. AC980390W
