Reversed-Phase Electrochromatography of Proteins on Modified

Anal. Chem. 1999, 71, 1621-1627

Reversed-Phase Electrochromatography of Proteins on Modified Continuous Beds Using Normal-Flow and Counterflow Gradients. Theoretical and Practical Considerations Christer Ericson and Stellan Hjerte´n*

Department of Biochemistry, Biomedical Center, University of Uppsala, P.O. Box 576, S-751 23 Uppsala, Sweden

Most chromatographic methods, including capillary electrochromatography (CEC), require gradient elution for high resolution of proteins. The gradients used in the CEC experiments described herein were generated by an HPLC instrument and pumped past one end of the capillary column. Part of the gradient was at the same time transported into the capillary solely by electroendosmosis. Employing these gradients, positively charged proteins were separated on a column filled with a continuous bed derivatized with C18 groups (for reversed-phase separation) and with ammonium groups (for generation of electroendosmotic flow (EOF)). Both the proteins and the EOF-generating ligands thus had positive charges to eliminate electrostatic interactions. The gradient and the sample were introduced at the same end of the capillary as in conventional (electro)chromatography or in a new approach, at different ends. In the former mode, the electroendosmotic velocity must be higher than the electrophoretic velocity, whereas in the latter mode, it must be lower. Accordingly, gradient elution in electrochromatography can be used for many CEC columns since the magnitude of their EOF is not critical. The EOF is a function of the concentration of the gradient constituents and may, therefore, be different in different segments of the capillary. The possible attendant effects on zone broadening have been treated, as well as the electrophoretic zone broadening and zone sharpening caused by the gradient. Special precaution was taken in order to ensure that the electrophoretic contribution to the recorded separation did not dominate over the chromatographic one. We used a new approach to synthesize continuous beds with ligands of high concentration. It can briefly be described as follows. By a suspension-polymerization off-capillary procedure (in the absence of stabilizers and surfactants), very small gel particles derivatized with C18 ligands are prepared under ultrasonication for 45 min. Then, piperazine diacrylamide (cross-linker) and dimethyl diallylammonium chloride (both EOF-generating ligand and cross-linker) are added. This suspension is propelled into the capillary (with a methacryloyl-activated inner surface). At this stage, the concentration of nonterminated polymer chains on the surface of the gel particles is sufficiently high for further polymerization reactions. The polymer bed 10.1021/ac9811470 CCC: $18.00 Published on Web 03/06/1999

© 1999 American Chemical Society

becomes attached covalently to the capillary wall concomitantly with the formation of channels in the bed. Synge and Tiselius were the first to use electroendosmosis to transport the mobile phase and the analytes in a chromatographic experiment, aimed at separating neutral degradation products of amylose by molecular sieving in an agar-agar jelly.1,2 The difficulties to get a high electroendosmotic flow in a gel and to derivatize native polysaccharides with ligands without destroying their gel-forming properties and to prepare synthetic gels with high UV transmission are some of the reasons why the approach of these Nobel laureates has not been considered until recently.3 Meanwhile, since 1974, electroendosmosis has been employed to propel liquid through beds packed with beads.4,5 The obvious disadvantages of the latter technique are (1) the difficulty to pack 10-50-µm capillaries uniformly with beads (that narrow capillaries should be employed to suppress efficiently thermal zone broadening at the high field strengths required for short analysis times) and (2) the zone broadening and bubble formation occurring at the frit supporting the bed. Both of these drawbacks were eliminated when continuous beds (monoliths) for capillary electrochromatography (CEC) were introduced.6-21 The separation * Corresponding author: (tel) +46 18 471 4461; (fax) +46 18 55 21 39; (e-mail) [email protected]. (1) Synge, R. L. M. Discuss. Faraday Soc. 1949, 7, 164-168. (2) Tiselius, A. Discuss. Faraday Soc. 1949, 7, 7-11. (3) Hjerte´n, S.; Ve´gva´ri, AÄ .; Srichaiyo, T.; Zhang, H.-X.; Ericson, C.; Eaker, D. J. Capillary Electrophor. in press. (4) Pretorius, V.; Hopskins, B. J.; Schieke, J. D. J. Chromatogr. 1974, 99, 2330. (5) Jorgenson, J. W.; Luckas, K. D. J. Chromatogr. 1981, 218, 209-216. (6) Hjerte´n, S.; Eaker, D.; Elenbring, K.; Ericson, C.; Kubo, K.; Liao, J.-L.; Zeng, C.-M.; Lidstro¨m, P.-A.; Lindh, C.; Palm, A.; Srichiayo, T.; Valtcheva, L.; Zhang, R. Jpn. J. Electrophor. 1995, 39, 105-118. (7) Liao, J.-L.; Chen, N.; Ericson, C.; Hjerte´n, S. Anal. Chem. 1996, 68, 34683472. (8) Ericson, C.; Liao, J.-L.; Nakazato, K.; Hjerte´n, S. J. Chromatogr., A 1997, 767, 33-41. (9) Fields, S. M. Anal. Chem. 1996, 68, 2709-2712. (10) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (11) Minakushi, H.; Nakanashi, K.; Soga, N.; Ishizuka, N.; Tanaka. N. J. Chromatogr., A 1997, 762, 135-146. (12) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (13) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288-2295. (14) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2296-2302.

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of low-molecular-weight compounds by CEC, particularly neutral ones, is a straightforward procedure and has, therefore, become very popular. The situation is quite different and there are many problems to solve when the sample consists of macromolecules. For example, due to their many binding sites, proteins most often require gradient elution to be separated with high resolution, although isocratic elution is possible at very low ligand densities.22 The main objectives of this study were as follows: (1) to find experimental conditions such that the gradient can be driven through the column solely by electroendosmosis,23,24,25 (2) to ensure that the inevitable electrophoretic separation of charged proteins does not dominate over the desired chromatographic separation based on partition in reversed-phase electrochromatography experiments; (3) to investigate whether the chromatographic zone sharpening that all concentration gradients create is attended by significant zone broadening caused by possible hydrodynamic flows originating from differences in electroendosmotic flow in different segments of the column; (4) to investigate whether the chromatographic zone sharpening caused by the gradient is counteracted or reinforced by electrophoretic zone broadening or zone sharpening caused by differences in electrophoretic velocities at the rear and at the front of a zone migrating in the gradient. EXPERIMENTAL SECTION Materials. Stearyl methacrylate, cytochrome c, ribonuclease A, lysozyme, and R-chymotrypsinogen were purchased from Aldrich (Steinheim, Germany). Piperazinediacrylamide, N,N,N′,N′tetramethylethylenediamine (TEMED), ammonium persulfate (electrophoresis purity reagents), and ammonium sulfate (HPLC grade) were from Bio-Rad (Richmond, CA). Methacrylamide and (3-methacryloyloxypropyl)trimethoxysilane (Bind-Silane A 174) were obtained from Fluka (Buchs, Switzerland). Dimethyldiallylammonium chloride was from Polysciences Inc. (Warrington, PA). Fused-silica capillaries (25- and 50-µm i.d., 365-µm o.d.) were from MicroQuartz (Munich, Germany). The mobile phase was passed through a filter with 0.20-µm pore size (Minisart RC 25, from Sartorius, Go¨ttingen, Germany) and then purged with nitrogen to expel dissolved oxygen. Preparation of Homogenized Continuous Beds for CEC. A high-intensity ultrasonic processor (600 W) fitted with a titanium alloy probe of 3-mm o.d. (Sonics and Materials Inc., Danbury, CT) was used for emulsification of the non-water-soluble stearyl methacrylate in an aqueous medium containing the water-soluble monomers. Sonication provides rapid and efficient homogenization of immiscible phases affording a very large interliquid surface contact area for the polymerization reaction. The power-ultrasound (15) Fujimoto, C. Anal. Chem. 1995, 67, 2050-2053. (16) Fujimoto, C.; Kino, J.; Sawada, H. J. Chromatogr., A 1995, 716, 107-113. (17) Schweitz, L.; Andersson, L.; Nilsson, S. Anal. Chem. 1997, 69, 1179-1183. (18) Nilsson, S.; Schweitz, L.; Petersson, M. Electrophoresis 1997, 18, 884-890. (19) Palm, A.; Novotny, M. V. Anal. Chem. 1997, 69, 4499-4507 (20) Fujimoto, C.; Fujise, Y.; Matsuzawa, E. Anal. Chem. 1996, 68, 3468-3472. (21) Asiaie, R.; Huang, X.; Farnan, D.; Horva´th, C. J. Chromatogr., A 1998, 806, 251-263. (22) Yao, K.; Hjerte´n, S. J. Chromatogr. 1987, 385, 87-98. (23) Huber, C. G.; Choudhary, G.; Horva´th, C. Anal. Chem. 1997, 69, 44294436. (24) Yan, C.; Dadoo, R.; Zare, R. N.; Rakestraw, D. J.; Anex, D. S. Anal. Chem. 1996, 68, 2726-2730. (25) Lister, A. S.; Rimmer, C. A.; Dorsey, J. G. J. Chromatogr., A 1998, 828, 105-112.

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influences the chemical interactions in the fluid through the welldocumented effect known as cavitation, which is the basis of sonochemical action. Two types of continuous-bed columns, a “moderate-EOF column” and a “high-EOF column” (see Elution Gradients), both derivatized with stearyl methacrylate for chromatography in the reversed-phase mode and dimethyldiallylammonium chloride for the generation of electroendosmosis, were prepared as follows. Piperazinediacrylamide (0.12 g) and methacrylamide (0.07 g) were dissolved in 1.6 mL of a sodium phosphate buffer (50 mM, pH 7.0). A 200-µL volume of this solution was transferred to a thick-walled conical glass test tube (9-mm i.d. at the top). Dimethylformamide (40 µL) and 160 µL (141 mg) of melted stearyl methacrylate (mp 18-20 °C) were then added. The polymerization was initiated by 20 µL of a 10% (w/v) aqueous solution of ammonium persulfate and 20 µL of 5% (v/v) TEMED. The sonication probe, set at 40% amplitude, was immersed in the monomer mixture (without touching the walls of the test tube to prevent glass splitter from being detached). The mixture was kept as an emulsion by continuous sonication for 40 min to obtain a stable dispersion of minute polymer particles. (In later experiments we added the stearyl methacrylate gradually during the first 20 min of the emulsification procedure.) Heat is generated during the emulsification (the temperature rises to about 70 °C following sonication for 10 min), which increases the reaction rate between the non-water-soluble stearyl methacrylate and the watersoluble monomers. There is no need for degassing by purging with helium or nitrogen, since the solution is instantaneously degassed by the power-ultrasound. Following addition of 300 µL of distilled water and 2 µL of an 1% (w/v) aqueous solution of potassium ferricyanide, the dispersion was immersed in a water bath of 20 °C and sonicated for an additional 5 min. (Potassium ferricyanide is an inhibitor of free-radical polymerization and was used to retard the reaction between the polymer particles which otherwise could form large aggregates before the emulsion was pressed into the capillary for the second “cross-linking” polymerization step). At this stage in the polymerization procedure, the concentration of nonterminated polymer chains on the surface of the polymer particles was still sufficiently high for the next reaction step, i.e., the in situ polymerization in the capillary. To 200 µL of the homogeneous emulsion was added 8 µL of dimethyldiallylammonium chloride for the moderate-EOF column (or 75 µL for the high-EOF column) and 20 µL of a solution of piperazinediacrylamide (0.6 g/mL in 50 mM sodium phosphate buffer, pH 7.0). The polymerization was reinitiated by adding 12 µL of a 10% (w/v) aqueous solution of ammonium persulfate and 15 µL of a 5% (v/v) aqueous solution of TEMED (30 µL for the high-EOF column). With the aid of a Plexiglas chamber pressurized with nitrogen, the emulsified reaction mixture was propelled into a fused-silica capillary, the walls of which had been pretreated with (3-methacryloyloxypropyl)trimethoxysilane.8 In this step, the living groups on the surface of the nonporous particles reacted with the piperazinediacrylamide, dimethyldiallylammonium chloride, and the methacryloyl groups at the capillary wall, thus forming a continuous polymer bed covalently attached to the wall, thereby eliminating the need for a supporting frit. To obtain a bed with the desired chromatographic properties, the emulsion in the capillary should form a rigid bed within 30 min, which can

be determined by visual inspection of the emulsion in the test tube. It may, therefore, be necessary to adjust the amount of TEMED. Finally, after 12 h, the column was washed with 10 void volumes of water and equilibrated with 5 mM sodium phosphate buffer, pH 2.0, containing 5% acetonitrile. A window for on-line detection was made in the column bed, as described previously.8 Dimethylformamide has several functions. It is compatible with the water-based free-radical copolymerization. Its concentration may be sufficient to enhance the reaction rate between the hydrophilic and hydrophobic monomers and facilitate the cavitation process during the sonication by lowering the surface tension. However, it cannot solubilize by itself the large amount of stearyl methacrylate used, nor can any detergent. The sonication step is, therefore, very important. Instrumentation. The equipment used for both capillary chromatography and electrochromatography in the gradient reversed-phase mode consisted of a gradient system for HPLC (models 2152 and 2150 from LKB, Stockholm, Sweden) which was connected to an extensively modified version of a previously described electrochromatographic system8 based on a CE apparatus (model HPE 100 from Bio-Rad Laboratories, Hercules, CA). A schematic diagram of the setup is shown in Figure 1. A reconstructed four-port microvalve (P451) from Upchurch (Oak Harbor, WA) served as a grounded electrolyte vessel, V1, through which the gradient passed. A PTFE membrane with 0.2-µm pore size from Millipore (Bedford, MA) was used to prevent gas bubbles formed at the platinum electrode from entering the vessel. The flow rate and back pressure were regulated by a adjustable microsplitter valve (P460S) SV, also from Upchurch. The splitting valve was used only for capillary chromatography and for washing the columns. A stainless steel six-port rotary valve, RV, from Rheodyne (Berkely, CA) was used for sample injection. A power supply with negative ground was from Glassman (0-30 kV, Whitehouse Stadium, NJ). BioFocus 3000 from Bio-Rad Laboratories was employed for the free-zone electrophoresis experiments. Elution Gradients. Two kinds of CEC columns were synthesized, one with moderate and one with high electroendosmotic flow. On the moderate-EOF column, the direction of the EOF was opposite to that of the separation, whereas on the high-EOF column, the direction was the same (as the hydrodynamic flow in conventional HPLC). 1. Counterflow Gradient for the Moderate-EOF Column. The counterflow gradient was created as follows. The HPLC pump with the LC controller was connected to the electrolyte vessel V1 via port P2 (Figure 1) and set to deliver a gradient at low flow rate and at zero pressure (the splitting valve SV was fully open). When voltage was applied, the gradient was propelled through the capillary solely by electroendosmosis. The buffer, 5 mM sodium phosphate, pH 2.0, contained 5% acetonitrile (A) and 80% acetonitrile (B). Both solutions were adjusted to have an apparent pH 2.0. The gradient went from 0 to 100% B in 25 min. Since the pressure in the electrolyte vessel V1 is zero the flow rate in not critical, but typical values were 0.2-0.3 mL/min. Trifluoroacetic acid, a common additive in RP-HPLC, was not used since it caused current drop during the runs. Injection of the sample was done electrokinetically at the anode for 20 s at 2 kV. Upon desorption, the protein migrated rapidly toward the cathode for on-tube detection since the electrophoretic velocity was larger

Figure 1. Schematic diagram of the setup used for gradient electrochromatography (“counterflow gradients” and “normal-flow gradients”) and gradient capillary chromatography. Depending on the gradient mode, either a high-EOF column, H, or a moderate-EOF column, M, was used. The arrows over the columns indicate the direction of the migration of the sample. PM, PTFE membrane. The polarity of the electric field was the same for both gradient modes. The splitting valve, SV, was used only for capillary HPLC and for washing the columns.

Figure 2. Diagram illustrating the principle for counterflow (a) and normal-flow (b) gradients. Observe that the direction of electroendosmotic flow is opposite to that of the electrophoretic movement in both methods and is opposite to the net migration velocity, vmigr ()|veo - velph|), in (a) and coincides with the net migration direction in (b). veo is not constant along the capillary, and velph is higher in the direction of the electrophoretic migration.

than the electroendosmotic velocity (Figure 2). In gradient version 2 described below, it was smaller. The column was prepared to give a net migration velocity of about 3 cm/min at 700 V/cm for a sample of cytochrome c (velph(true) ) 4.5 cm/min and veo ) 1.5 cm/min, giving velph(apparent) ) 3.0 cm/min). See Figure 3a. 2. Normal-Flow Gradient for the High-EOF Column. The column was synthesized to produce a net migration rate of approximately 3 cm/min at 700 V/cm (velph(true) ) 4.5 cm/min Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

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Figure 3. Chromatograms obtained by gradient electrochromatography (a, b) and reversed-phase chromatography (c). Sample: ribonuclease A (R); cytochrome c (C); lysozyme (L); R-chymotrypsinogen (Ch). Protein concentration: 0.6 mg/mL of each protein except for ribonuclease A, which had a 3-fold higher concentration. Mobile phase in all runs: a linear gradient from 5 to 80% acetonitrile in 5 mM sodium phosphate, adjusted to pH 2.0. On-tube detection at 280 nm. Columns: 8.0 cm (6 cm effective length) × 50 µm i.d. × 375 µm o.d. filled with ultrasonically homogenized continuous beds derivatized with octadecyl groups and two different concentrations of ammonium groups to obtain columns with different EOF. (a) moderate-EOF column and (b) high-EOF column. In (a) and (b), the samples were eluted by electroendosmotically driven gradients. In (a), the running direction of the gradient (determined by the direction of EOF) was opposite to the migration direction of the sample (counterflow gradient (Figure 2a)); in (b), the gradient and sample had the same direction (normal-flow gradient (Figure 2b)). In the counterflow mode, the gradient reached the window, w, and the sample, s, after 2 and 6 min, respectively. Applied voltage, 5.5 kV (700 V/cm); (c) conventional capillary RP-HPLC; pressure, 50 bar. A comparison of the separation patterns a-c indicates that it is possible to design almost “clean” CEC reversed-phase experiments, i.e., with only small contributions from the attendant electrophoresis (see also Figure 4.).

and veo ) 7.2 cm/min, giving velph(apparent) ) 2.7 cm/min). The six-port rotary valve RV was used to transfer the sample to the electrolyte vessel V1. The flow was then stopped for electrokinetic injection of the sample. Using the same polarity of the voltage, the normal-flow gradient was formed in the same way as the counterflow gradient. However, the separation direction was the opposite, since the electroendosmotic velocity was larger than the electrophoretic velocity. The positively charged proteins thus moved from the cathode to the anode (see Figures 2b and 3b). 3. Gradient Elution in Pressure-Driven Capillary Chromatography. The protein sample was eluted under pressure by the gradient described in section 1 using the moderate-EOF column (effective length 6 cm). In this experiment (Figure 3c), the adjustable splitting valve (SV in Figure 1) was employed to achieve the low flow rate required. Sample injection was done electrokinetically as described in section 2. 4. “Plain” electrophoretic runs on the continuous beds were performed in 5 mM sodium phosphate, pH 2.0, containing 80% acetonitrile (Figure 4a). The proteins do not interact with the bed at this high concentration of acetonitrile. A free-zone electrophoresis was performed in this buffer but without addition of acetonitrile (Figure 4b). 1624 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

Figure 4. Electropherograms of the proteins used in Figure 3. (a) Capillary electrophoresis using the continuous bed column described in (b). Mobile phase, 5 mM sodium phosphate containing 80% acetonitrile, pH 2.0 (apparent); the proteins do not interact with the bed at this acetonitrile concentration. Sample, see the legend to Figure 3; applied voltage, 4 kV (500 V/cm). The effective electrophoretic separation distance was calculated at about 9.0 cm considering that the EOF counterflow was 1.2 cm/min at 500 V/cm. This electropherogram mimics the electrophoretic contribution in the chromatograms in Figure 3a and b. (b) Free zone capillary electrophoresis. Column, 30 cm (25 cm effective length) × 25 µm i.d. × 375 µm o.d., coated with linear polyacrylamide to eliminate electroendosmosis; buffer, 5 mM sodium phosphate, pH 2.0; applied voltage, 3 kV (100 V/cm). Other conditions as in Figure 3.

RESULTS AND DISCUSSION Determination of Relative Electrophoretic and Electroendosmotic Velocities in Different Cross Sections of the CEC Column upon Gradient Elution. Since the buffer composition varies along the capillary column in gradient runs, one should consider the possibility that the electroendosmotic velocity also varies, which could cause peak distortion (see Factors Affecting the Width of a Zone upon Gradient Elution in CEC). Therefore, we have determined the relative velocities in some cross sections of the capillary from experimentally determined ueo and κ-values, using the expression

veo ) ueo(i/κ)

(1)

where veo and ueo are the electroendosmotic velocity and mobility, respectively, κ is the conductivity, and i is the current density. To mimic the composition of the gradient employed in the experiments presented in Figures 3, six buffer solutions with different acetonitrile concentrations were prepared by mixing two 5 mM sodium phosphate buffers, one containing 5% (A) and one 90% (B) acetonitrile, both adjusted to pH 2.0 with phosphoric acid. The electroendosmotic velocity in a moderate-EOF column was determined for each of the six buffer solutions with acetone as an unretained, neutral, marker. The electroendosmotic mobilities (ueo) calculated from the field strength (700 V/cm) were plotted against the acetonitrile concentration (Figure 5a). Using these ueo values and the measured κ-values of the above six mobile phase solutions, the relative veo values (veo,rel) were calculated from eq 1 for six cross sections of the column (Figure 5a). Observe that the phosphate concentration varies in the gradient, which means that no conclusions can be drawn about how the acetonitrile

a

b

Figure 5. (a) Electroendosmotic mobility (ueo) and relative electroendosmotic velocity (vrel,eo) as a function of the composition of the gradient used for elution of the proteins in the experiment presented in Figure 3. Alterations in the composition of the gradient are caused not only by alterations in the concentrations of acetonitrile but also by those of the phosphate buffer. Column, 8.0 cm (6.0 cm effective length) × 50 µm i.d. × 375 µm o.d. filled with homogenized continuous bed derivatized with ammonium and octadecyl groups (moderateEOF column); mobile phase, 5 mM sodium phosphate with increasing concentration of acetonitrile, pH 2.0 (adjusted); sample, acetone. For calculation of vrel,eo, see text. (b) Electrophoretic mobility (uelph) and relative electrophoretic mobility (vrel,elph) as a function of the composition of the gradient used for elution of the proteins in the experiments presented in Figure 3. Sample, lysozyme; column, 25 cm (20 cm effective length) × 25 µm i.d. × 375 µm o.d., coated with linear polyacrylamide to eliminate electroendosmosis; mobile phase, see (a).

concentration alone affects ueo. Since the current density (i) has the same value in all cross sections of the capillary at a given moment, i is canceled in the calculation of relative electroendosmotic velocities. The relative electrophoretic velocities, vrel,elph, were also calculated from determinations of electrophoretic mobilities of a protein (lysozyme) by free zone electrophoresis in five phosphate solutions of different acetonitrile concentrations and conductivities (Figure 5b). The approach was analogous to that described above for the determination of relative electroendosmotic velocities. The equation u ) 0rζ/η applies for both the electroendosmotic and the electrophoretic mobilities (ζ is the zeta potential of the continuous bed and lysozyme, respectively, in the experiments described). It is, therefore, not surprising that veo,rel in Figure 5a and velph,rel in Figure 5b are affected similarly by the acetonitrile concentration and the conductivity. Factors Affecting the Width of a Zone upon Gradient Elution in CEC. 1. Chromatographic Zone Sharpening. The acetonitrile gradient, like all desorbing gradients used in chromatography, affords narrow zones when designed properly. The

ideal composition and the slope of the gradient should be such that the analytes are not completely desorbed until they leave the column. Otherwise, the analytes migrate for some time without interaction with the bed, i.e., under zone broadening. 2. Electrophoretic Zone Sharpening and Zone Broadening. Zone sharpening occurs when the field strength and, thereby, the electrophoretic velocity at the rear of the zone is larger than at its front and zone broadening occurs in the opposite situation (“rear” and “front” here refer to the electrophoretic migration direction). As shown in Figure 5b, the relative electrophoretic velocity in different sections of the column increases when the acetonitrile concentration increases, which is equivalent to zone broadening (see Figure 2). To obtain the same vrel,elph on both sides of the migrating analyte zone, and consequently no zone broadening (or preferably a lower relative velocity at the higher acetonitrile concentrations to achieve electrophoretic zone sharpening), the ionic strength or the viscosity must be higher in the buffers containing the higher acetonitrile concentration. But these measures have the large drawback that the electroendosmotic flow will decrease during the course of a run due to the high conductivity (and/or viscosity) of the mobile phase. However, the electrophoretic zone broadening appears to be negligible, compared to the zone-sharpening effect of the gradient itself (compare parts a and b of Figure 3 with part c). 3. Speculations on Zone Broadening Caused by Different Electroendosmotic Velocities in the Gradient. The net flow velocity is the same in all cross sections along the capillary. Accordingly, differences in electroendosmotic velocities generate hydrodynamic flows in the form of rotation of the mobile phase in the voids between adjacent gel particles or/and a stream toward the end(s) of the capillary.26-30 Presumably, the rotational flow will be predominant, since the flow resistance to transport liquid in an axial direction from void to void is larger than that required to cause a liquid to rotate in a void. If this assumption is correct, the zone broadening caused by differences in EOF should be negligible (at least if the differences in EOF are relatively small). Interestingly, a liquid rotation could even favor the resolution, since an analyte would be transported faster from one gel particle to another by this rotation than by diffusion alone. Besides the above zone broadening, one may imagine a zone broadening induced by a pressure gradient generating a parabolic zonal velocity profile. Do the Conceivable Differences in Electroendosmotic Velocities in a CEC Column Cause Demonstrable Zone Broadening? A continuous change in EOF in the capillary during the course of a run could give rise to zone broadening (see the above section 3). In an experimental investigation of this broadening, a sample of acetone was eluted by EOF both isocratically (80 and 5% acetonitrile) and by a gradient running from 5 to 80% acetonitrile in 4, 10, and 25 min. Acetone was injected both in front of the gradient and when half of the gradient had eluted. We used a continuous bed which had been derivatized with ammonium groups (the moderate-EOF column; see above). The C18 ligands were omitted in order to get a flow pattern (26) O ¨ fverstedt, L.-G.; Johansson, G.; Fro¨man, G.; Hjerte´n, S. Electrophoresis 1981, 2, 168-173. (27) Chien, R.-L.; Helmer, J. C. Anal. Chem. 1991, 63, 1354-1361. (28) Hjerte´n, S.; Kubo, K. Electrophoresis 1993, 14, 390-395. (29) Potocek, B.; Gas, B.; Kenndler, E.; Stedry, M. J. Chromatogr., A 1995, 709, 51-52. (30) Gas, B.; Stedry, M.; Kenndler, E. J. Chromatogr., A 1995, 709, 63-68.

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Figure 6. Comparison of acetone peaks obtained by gradient CEC and isocratic CEC. Column, moderate EOF-column as in Figure 3a but without C18 ligands. Isocratic electroendosmotic elution in 5 (a) and 80% (b) acetonitrile. Using an electroendosmotically driven gradient running from 5 to 80% acetonitrile in 4 min, acetone was injected both in front of (c) and when half of the gradient had eluted (d). Observe that similar peak widths were obtained in the different elution modes; 10- and 25-min gradients gave similar peak shapes.

unaffected by hydrophobic interaction with the bed. Acetone, one of the most common neutral markers, was assumed to migrate with the same velocity as the mobile phase. Therefore, the gradient itself should not cause any zone sharpening. A possible distortion of the acetone zone by EOF differences should be stronger the steeper the gradient. Therefore, the time for one of the chosen gradients was only 4 min, corresponding to a volume approximately equal to the dead volume of the bed. Interestingly, no significant differences in zone broadening and peak shapes were observed when the experiments were performed in the gradient and isocratic mode (Figure 6) and the plate numbers were similar (240 000-265 000 N/m). We therefore concluded that a slow and monotonic change in electroendosmosis caused by the elution gradient did not significantly distort the sample zone. However, the zone deformation may be significant when abrupt changes in EOF occur (e.g., at a gap in the bed).26-30 Design of CEC Beds Compatible with Gradient Elution of Proteins. The highest selectivity for the separation of polypeptides and proteins by reversed-phase chromatography is often obtained when the pH of the buffer is below the pI of the proteins. At such a pH, many proteins have a large relative retention, R; i.e., the ionization of the carboxyl groups is suppressed and the amino groups are fully protonated. Accordingly, the proteins will behave as positively charged ions and move electrophoretically in the same direction in CEC experiments. One of the largest difficulties in this study was to design beds appropriate for gradient elution of macromolecules and find experimental conditions that suppress the electrophoretic separation. A bed for separation of proteins by RP-CEC must be derivatized not only with nonpolar ligands but also with charged ligands to generate electroendosmosis. To avoid any electrostatic interaction with the proteins, the ligand should be positively charged when the run is done at low pH. We have found that it is extremely difficult to find conditions that permit high resolution in RP-CEC of macromolecules having a charge opposite to that of the ligands, i.e., when electrostatic interaction is superimposed on partition, particularly since a low density of charged ligands cannot be used in CEC because of the attendant low EOF. One approach was to introduce moderate amounts of positively charged ligands into the bed and drive the gradient “backward” as a counterflow; i.e., the direction of EOF should be 1626 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

opposite to that of the separation (see Elution Gradients and Figures 2 and 3a). In another approach, the proteins were eluted in a more straightforward way in a direction coinciding with the separation direction (normal-flow gradient; see Figures 2 and 3b) which requires an electroendosmotic velocity much higher than the electrophoretic velocity of the proteins. The concentration of the charged monomer (ligand) must be high for this requirement to be fulfilled. However, differences in reaction rates between the charged and nonpolar ligand in the synthesis of the continuous bed may lead to a low density of the nonpolar ligand or make the polymer matrix brittle. Fortunately, using the synthesis method described herein, whereby the charged monomer was coupled in a second step to minute polymer particles in an emulsion, we could achieve an electroendosmotic velocity that was almost twice the electrophoretic velocity for cytochrome c and yet accomplish a high concentration of stearyl methacrylate, the nonpolar ligand. As mentioned, all attempts to introduce low concentrations of negatively charged ligands (e.g., SO3- groups) in the bed and separate the proteins below their pI were unsuccessful, due to the inevitable electrostatic interactions with the proteins. These interactions can be suppressed by adding salt to the mobile phase, but at the expense of the electroendosmotic velocity. Moreover, at these high ionic strengths, the Joule heat may cause zone deformation and gas bubbles, especially if the capillary is not cooled (this broadening can, however, be strongly suppressed if capillaries with diameters of e25 µm or/and low-conductivity buffers are used31). Instead of using a pump to generate the gradient, one can aspirate manually an array of buffer segments32-34 into the PEEK tubing connected to port P2 in Figure 1 and deliver this gradient, without splitting, to the electrode vessel V1 by a syringe pump or a thermal expansion pump.33 Comparison between Gradient Elution Chromatograms in RP-CEC and RP-HPLC. The model proteins were desorbed from both the moderate-EOF column and the high-EOF column by a 25-min linear gradient (5-80% acetonitrile). It should be noticed that, following desorption, the proteins migrated by electrophoresis for less than 2 min before detection, which means that the contribution of electrophoresis to the net separation was small. The resolution and the order of the peaks in both modes of gradient electrochromatography and gradient RP capillary chromatography were similar (Figure 3), which is an experimental indication (1) that the separations obtained in the CEC runs were essentially based on reversed-phase chromatography rather than electrophoresis, (2) that the two gradient modes give similar separations, and (3) that capillary chromatography of proteins on the continuous beds exhibits the same high performance as does CEC. Zone Electrophoresis of the Model Proteins on the Moderate-EOF Column To Demonstrate Further the Small Electrophoretic Contribution to the Separations in CEC. The proteins were separated isocratically by electrophoresis on the (31) Hjerte´n, S.; Valcheva, L.; Elenbring, K.; Liao, J.-L. Electrophoresis 1995, 16, 584-599. (32) Ericson, C.; Hjerte´n, S. Anal. Chem. 1998, 70, 366-372. (33) Li, Y.-M.; Liao, J.-L.; Nakazato, K.; Mohammad, J.; Terenius, L.; Hjerte´n, S. Anal. Biochem. 1994, 223, 153-158. (34) Liao, J.-L.; Li, Y.-M.; Hjerte´n, S. Anal. Biochem. 1996, 234, 27-30.

moderate-EOF column with 80% acetonitrile in the buffer with a low electroendosmotic counter flow of 1.1 cm/min at 500 V/cm (which gave an effective electrophoretic separation distance of about 9 cm). As expected, the separation was poor on such a short column(Figure 4a). However, the proteins were well separated by carrier-free zone electrophoresis using a longer column (25 cm) which had been coated with linear polyacrylamide to eliminate EOF (Figure 4b). The migration order was quite different from that obtained by gradient RP-CEC (Figure 3a and b), which is another indication that the separations in gradient RP-CEC are predominantly based on partition and not electrophoresis. However, a closer comparison of the HPLC and CEC chromatograms reveals some contribution from electrophoresis. For instance, R-chymotrypsinogen (low velph) elutes relatively late in the CEC experiments (especially using the counterflow gradient). The maximum difference in relative elution time for R-chymotrypsinogen and lysozyme in the CEC and RP-HPLC experiments is 14%. Although the combination electrophoresis/electrochromatography may sometimes give higher resolution than electrophoresis and chromatography do separately, it should be emphasized that the difficulty in getting reproducible results always increases when a method is based on more than one separation mechanism (high reproducibility requires that the contributions from both separation mechanisms are constant during a series of experiments). Advantages of the Continuous Beds in CEC Experiments. Continuous beds for electrochromatography provide several advantages. The beds are relatively easy to prepare, even in capillaries with inner diameters well below 25 µm,32 since the capillaries are not packed with beads as in conventional columns but filled with a solution (or an emulsion of polymer particles as described herein) which is then polymerized in situ. The beds can be synthesized to give relatively low back pressures while retaining high efficiency or, alternatively, with high back pressures (small pores) and still higher efficiency. The magnitude and direction of the electroendosmosis can readily be tuned to the desired application by altering the concentration or the type of the EOF-generating charged ligand. In an isocratic elution the ζ-potential and, thereby, the electroendosmosis, is the same in any cross section of the column since the polymerization conditions are identical in all sections of the capillary. The risk of bubble formation caused by differences in EOF in different segments of the column is, accordingly, small and it is, therefore, not necessary to pressurize the electrode vessels (nor in gradient elution, as our experiments indicate). By increasing the pressure in the adjustable splitting valve (SV in Figure 1), a hydrodynamic flow is generated through the column which can be used alone for elution in capillary chromatography, in combination with the electroendosmotic flow in a CEC run,35 or for washing the column. The simple equipment in Figure 1 is thus characterized by flexibility and versatility. The continuous bed is covalently attached to the wall of the capillary to eliminate the need for a supporting frit, which has several advantages.6-8,32-34 In CEC, the polyimide layer is often stripped off at the ends of the capillary when the buffer contains organic solvents (such as acetonitrile) with the inevitable result

that the capillary ends become brittle and are easily broken. Any packed column with frits is then useless. The frit-free continuous beds can still be employed after the damaged end has been cut off. Notice that the two gradient elution modes described herein are not restricted to operate on continuous polymer beds. In fact, any type of positively charged reversed-phase bed may be utilized, provided that the ratio between EOF and electrophoresis can be tuned. We achieved this tuning in a straightforward way by altering the amount of charged ligands on the surface of the bed. But other methods may also be successful. For instance, buffer constituents forming ion pairs with the ligands or surfactants below their cmc may alter both the partition coefficient and EOF.7

(35) Behnke, B.; Bayer, E. J. Chromatogr., A 1994, 680, 93-98.

AC9811470

CONCLUSIONS 1. Charged proteins can now be separated by CEC in the reversed-phase mode using gradient elution with negligible influence from electrophoretic migration. 2. The separation profiles and the resolution are similar in CEC and in RP-HPLC on the same column with the same gradient propelled through the column by an HPLC pump, indicating that the electrophoretic contribution to the separation is small. 3. Two methods for gradient elution can be employed: (a) the conventional one, where the velocity of electroendosmosis veo is larger than the net migration velocity of the sample vmigr, or (b) a new one, where veo < vmigr. This flexibility as to the magnitude of veo is of particular interest to those who do not synthesize their own CEC columns and are therefore dependent on commercially available CEC columns of varying EOF. 4. In the new method for gradient elution (veo < vmigr), the gradient and the sample migrate in opposite directions; i.e., the effective electrophoretic migration distance is larger than the length of the column. This elution method has the advantage compared to the classical method (where the gradient and the sample migrate in the same direction) that shorter columns can be used without loss in resolution. Accordingly, lower voltage can be employed for a given field strength, which has the advantages that the risk of electrical shock is lower and that less expensive power supplies can be used. Short continuous beds are of particular interest for CEC on microchip-based devices. 5. Ultrasonically homogenized continuous beds derivatized with different ligands permit large variations in the degree of substitution. There are thus no practical limitations in the choice between the two above methods available for gradient elution. 6. Experiments analogous to those described for the analysis of positively charged proteins can be performed with negatively charged proteins (running at a pH higher than the protein pI) and negative ligands. RP-HPLC experiments are, however, seldom performed at such high pH values (>6-7). ACKNOWLEDGMENT This study has been supported financially by the Swedish Natural Science Research Council and the Swedish Research Council for Engineering Sciences. Received for review October 20, 1998. Accepted January 15, 1999.

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