Standard and Capillary Chromatography, Including

Ind. Eng. Chem. Res. 1999, 38, 1205-1214

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Standard and Capillary Chromatography, Including Electrochromatography, on Continuous Polymer Beds (Monoliths), Based on Water-Soluble Monomers Stellan Hjerte´ n* Department of Biochemistry, Uppsala University, Biomedical Center, P.O. Box 576, S-751 23 Uppsala, Sweden

Continuous polymer beds (acrylic-based beds) have the following advantages over beds packed with beads: (1) For most chromatographic modes they can be synthesized in one step by sucking a solution of appropriate monomers into a chromatographic tube and polymerizing it under such conditions that a polymer rod forms containing channels which permit hydrodynamic flow of the mobile phase at a relatively low back pressure. The expensive and time-consuming preparation of beads by the conventional suspension-polymerization procedure is thus omitted, as well as the packing step. (2) The monomers are water-soluble, which means that the polymer bed is biocompatible and that no organic solvents are required for the synthesis, which is attractive from an environmental and economical point of view (the destruction or regeneration of organic solvents often costs more than the purchase). (3) The bed can be prepared in situ as described above or, alternatively, by simple packing with bed material synthesized separately by the same procedure. The latter alternative permits a great number of columns to be packed from the same batch. (4) The rate of success of the preparation of beds is close to 100%. (5) The gel particles constituting the bed are nonporous to increase the mass transfer. Yet, the binding capacity of proteins is high because the area of the particles is large because of their rough surface and small diameters. (6) The gel particles are covalently linked, which gives a low flow resistance, notwithstanding that the gel particles are small (0.2-0.5 µm). (7) The bed can be compressed to decrease the distance between the gel particles and thereby increase the resolution (the method is not suitable for capillary columns, for which the monomer concentration is increased instead). The back pressure is low (often below 50 bar) even at high flow rates (100 cm/min). (8) Following compression of the bed, the resolution often is constant or even increases with an increase in flow rate, contrary to classical chromatographic theory. (9) No frit to support the bed is required in capillary chromatography, including electrochromatography, because the bed can be covalently attached to the capillary wall (a frit often disturbs the separation). (10) The recently introduced completely homogeneous continuous gel beds have the advantage that zone broadening caused by Eddy diffusion is eliminated. Introduction The first continuous support columns consisted of polyurethane foams cast in situ. They were intended for both gas (Ross and Jefferson, 1970; Schnecks and Bieber, 1971; Hileman et al., 1973) and liquid chromatography (Kubin et al., 1967; Ross and Jefferson, 1970; Lynn et al., 1974). Unfortunately, the chromatographic properties were not satisfactory, and two decades transpired before columns of this type, designed according to other principles, could compete favorably with packed beds (Hjerteń et al., 1989a; Svec and Frećhet, 1992). The synthesis is done in situ in the chromatographic tube under such conditions that the polymer rod formed is crisscrossed by intercommunicating channels through which the eluent can pass (alternatively, the bed material described herein can be synthesized off-tube). Principally, the preparation method is simple and has the great advantage that two cumbersome, expensive, and time-consuming steps can be omitted: the preparation of beads and the packing of the column. However, it is not an easy task to design a synthesis method which * Telephone: +46 18 4714461. Fax: +46 18 552139. Email: [email protected].

affords a bed with the desired properties, which is illustrated very well by the following quotation from a letter I received shortly after our first paper on the continuous beds was published (Hjerteń et al., 1989a): “These papers are of great interest to me: For years I have been considering ‘continuous’ beds, but Prof. XX and Dr YY warned me that the high resistance and slow flow-rate would be difficult to overcome.” My answer, based on the experiments described below, is, certainly, also illuminating.” As your colleagues have pointed out it is not easy to synthesize continuous beds with low flow resistance. I believe that many researchers have tried to prepare such beds, but the difficulties to achieve both high flow rates and high resolution are not so easy to overcome.” This problem is obvious in a paper by Kumakura et al. (1989). In 1959-1960, I tried to find gels for molecular-sieve electrophoresis with properties better than those of starch, which afforded a resolution far superior to that of any other gel medium available at that time (Smithies, 1962). Cross-linked polyacrylamide gels appeared to be an excellent substitute [two American groups headed by Ornstein and Raymond, respectively, made the same observation at the same time (Hjerteń, 1988a)]. Upon an increase in the concentration of the cross-

10.1021/ie970676b CCC: $18.00 © 1999 American Chemical Society Published on Web 03/19/1999

1206 Ind. Eng. Chem. Res., Vol. 38, No. 4, 1999

linker, N,N′-methylenebis(acrylamide), I observed that the gel became white and somewhat granular. Because I worked also in the field of chromatography, it was fairly obvious to me to investigate whether this gel, prepared directly in a chromatographic tube, could be used for molecular-sieve chromatography, the chromatographic counterpart of molecular-sieve electrophoresis. It turned out to be so: monomer and dimer of albumin could be separated. However, the flow rate was very low, and the experiments were, therefore, not continued. When a very able Ph.D. student, Jia-Li Liao, joined my group, I decided to resume the experiments. With important assistance from him and later from other guest researchers and doctoral candidates, a series of systematic studies were carried through with the aim to optimize the experimental conditions to obtain maximum resolution at high flow rates and low-to-moderate pressures. In this review I will discuss in general terms the preparation of the continuous beds and their properties in different chromatographic methods. Only such details will be given as illustrate what is characteristic of the beds. Other details can be found in the references given. The present article deals with supports synthesized from water-soluble monomers. In this series of reviews, beds based on water-insoluble monomers are also described (see Svec and Frećht, 1992). For continuous silica supports, see Fields (1996) and Minakuchi et al. (1996). There seems to be a general trend to change the original terminology of (separation) methods. A classical example is the term “Displacement Electrophoresis” (suggested by A. J. P. Martin, the Nobel Prize Winner in Chemistry in 1952), which is nowadays often exchanged for “Isotachophoresis”. In recognition of the pioneers, one should keep the notation they introduced if there are no particular reasons to change it (Hjerteń, 1973). Therefore, throughout the text in this article, I employ the term “continuous beds”, not “monoliths”. To What Extent Can the Zone Broadening in a Chromatographic Gel Bed Approach the Extremely Small Broadening Characteristic of Gel Electrophoresis? Continuous beds were introduced not only to avoid packing of columns with already prepared beads but also with the hope to create beds which give very little zone broadening (Hjerteń et al., 1989a). Let us, therefore, discuss whether the zones in chromatography can be made as narrow as those in gel electrophoresis: a method which, from this point of view, is ideal because the adsorption is extremely low and the gels are perfectly homogeneous macroscopically. When adsorption is negligible, the plate height (which is a measure of the zone width) in electrophoresis is determined by the width of the applied zone, diffusion, and Joule heating. The plate height observed in chromatography is governed by the same factors, although the last one can be neglected, except at extremely high flow rates. However, there are three additional factors which are of utmost importance: (1) the nonuniform flow caused by heterogeneity in the bed (giving rise to so-called Eddy diffusion), (2) the time taken for a solute to adsorb to the bed and desorb (on/off kinetics), and (3) the time needed to move from the mobile to the stationary phase and vice versa (the residence time in the mobile phase). No separation takes place as long as the solutes stay in

Figure 1. High-performance anion-exchange chromatography of proteins on a column packed with nonporous agarose beads at increasing flow rates and consequent decreasing void volumes achieved by compression of the bed (Hjerteń et al., 1988b). The decrease in peak width upon an increase in flow rate is caused by a decrease in void volume (see text) (and to some extent probably also to an increased degree of nonlaminar flow and perhaps also some contribution from the hydrodynamic “lift force”). This flow behavior for compressible agarose beds is characteristic also of continuous beds [and beds of coated silica beads (Hjerteń et al., 1991b]. Reproduced with permission of the publisher.

the mobile phase, whereas in ideal electrophoresis, the separations are accomplished only in the liquid corresponding to this phase. Accordingly, the resolution in electrophoresis is independent of the volume of this liquid, whereas in chromatography, it increases when the analogous volume () the volume of the mobile phase) decreases. I will now discuss how to minimize the effects of these factors on chromatographic zone broadening and make comparisons with the analogous electrophoretic parameters. In practice, it is not possible to prepare a chromatographic bed as uniform as an electrophoretic gel medium, even if all of the gel particles making up a packed or a continuous bed are quite identical. In addition, hydrodynamic flow through a chromatographic column requires that the average distance between the gel particles is considerably larger than the gel pores (channels) through which the proteins migrate upon electrophoresis (this is one reason why gel electrophoresis affords narrower zones than does chromatography). To minimize this distance, the diameters of the beads in a bed should be small, but the limit is set by the attendant increase in pressure drop. An important feature of capillary electrochromatography (see below) is that the size of the bed particles can be much smaller than those in regular chromatography, and this produces sharper peaks. Although the gel particles making up a continuous bed are much smaller (0.2-0.5 µm) than those used in a packed bed, the continuous beds nevertheless have the advantage over packed beds in that they can be compressed with only a moderate increase in flow resistance because the gel particles are linked by covalent bonds. Upon compression, the volume between the gel particles (the dead volume) decreases, which means less zone broadening because of the shorter residence time of the solutes in the mobile phase (see Figure 1). The residence time would be reduced further if the laminar flow could be replaced by a nonlaminar flow, which affords a much faster transport of a solute from one bead to another than does diffusion, which is a slow process (see “The Attractive Relationship be-

Ind. Eng. Chem. Res., Vol. 38, No. 4, 1999 1207

tween Resolution and Flow Rate for Continuous Beds”). Maybe, the on/off kinetics can be accelerated by this flow (and perhaps also by the hydrodynamic lift force) and, thereby, the mass transfer. From the above theoretical considerations, one can conclude that, at the present stage of the development of chromatography, the zones obtained are in most cases wider than electrophoretic zones, which is in accordance with practical experiments. However, an entirely homogeneous continuous bed can be synthesized if the cross-linker is omitted from the monomer solution (see next section). Another very recent alternative is described in “Completely Homogeneous Continuous Gel Beds”. These approaches open the possibility to obtain zones as narrow as those in electrophoresis. The continuous beds seem to have the advantage of being chromatographically more homogeneous than conventional packed beds (see the discussion of Figure 2VI in “Capillary Electrochromatography”). All of the above discussions refer to isocratic elution. If gradient elution is employed, the chromatographic zones may occasionally become as sharp as those in gel electrophoresis due to the focusing effect of the gradient. Separation in Non-Cross-Linked Continuous Beds, i.e., in Entirely Homogeneous Polymer Solutions Continuous beds are composed of cross-linked polymers to which ligands are coupled. Macromolecules interact almost exclusively with the ligands, whereas aromatic low-molecular-weight compounds may interact both with the ligands and the matrix, which is typical of most chromatographic supports [the separation patterns were the same when basic drugs were subjected to electrophoresis in non-cross-linked and cross-linked polyacrylamide (see parts h and i of Figure 10 in Hjerteń et al., 1989b)]. The non-cross-linked polymer (with or without ligands) dissolved in an appropriate solvent can, therefore, also be employed for separation, provided that the solutes and the polymers can be transported with different velocities and, as in all separation methods, the solutes interact differently with the polymer. It is quite irrelevant whether the velocity of the polymer is 0 [as is the case for the (cross-linked) stationary phases in chromatography] or differs from 0 (as may be the case in ultracentrifugation and electrophoresis). Observe that in a polymer solution differences in velocity between solutes and the polymer cannot be accomplished by a hydrodynamic-flow transport, only by a transport generated by centrifugal (ultracentrifugation) or electrical (electrophoresis, electroendosmosis) fields. The latter alternative has been used for the separation of acidic and basic substances in the presence of nonionic and ionic polymers (the analysis was performed in the CE mode) (S. Hjerteń et al., 1989b; Terabe and Isemura, 1990; Ozaki et al., 1995). When the solute is nonionic, the polymer must be ionic if an electrical field is employed to create the velocity difference. The electroendosmotic flow at the tube wall, often generated by a physical adsorption of the charged polymer onto the walls, displaces the polymer solution and can, therefore, affect the migration times but not the order of migration. The great advantage of a completely homogeneous polymer solution, compared to a heterogeneous chromatographic bed, is that the zone broadening is much smaller, provided that the on/off interaction of the solute

with the polymer is fast (the residence time in the “mobile phase” is short). Besides, a polymer solution can easily be removed from the separation tube following a run and be replaced by a fresh one, which gives high reproducibility and renders automation easier. There are only a few experiments reported in which non-crosslinked polymers are utilized to achieve separation or affect the separation pattern. No attempts at systematizing this approach have been made. The reason may be that we are often blocked by the great impact words (in this case, the terms chromatography and electrophoresis) have on our thoughts. In fact, it is a question of definition whether a separation in a polymer solution should be called electrophoresis or chromatography, because not only in chromatography are the separations based on interactions but also in many experiments in electrophoresis (for instance, complexation with protons, metal, and borate ions). The conventional chromatographic bed has a dual function: to achieve separation and to stabilize the zones against convection. A polymer solution cannot serve the latter function (the high viscosity can, however, decrease the sedimentation speed), and, therefore, special precautions must be taken to eliminate convection; for instance, the experiments can be conducted in a stationary or rotating narrow-bore tube for analytical runs (Hjerteń, 1967) or in a wider tube containing a density gradient for preparative runs (Svensson and Valmet, 1955). The above suggestion to base separations on noncross-linked polymers instead of conventional chromatographic beds will, no doubt, work excellently without practical difficulties for isocratic experiments with lowmolecular-weight compounds. The method also permits similar experiments with macromolecules, provided that the ligand density is low (Yao and Hjerteń, 1987). However, most chromatographic experiments are performed in the gradient elution mode. The corresponding experiments in a polymer solution have not yet been done. Many difficulties can be foreseen when high salt concentrations are required for desorption, because of the attendant low electrophoretic velocity and electroendosmotic flow and high Joule heat. These and other problems must be overcome before the method can also become applicable for gradient elution of macromolecules. However, the gradient method employed for reversed-phase separation of proteins by capillary electrochromatography on continuous beds can probably be applied also to analogous gradient separations in solutions of (neutral) polymers [see “Capillary Electrochromatography (CEC)”]. Chromatograms Illustrating the Multitude of Chromatographic Modes Continuous Beds Offer for Both Standard and Capillary Chromatography Continuous beds have been employed for anion- and cation-exchange chromatography (Hjerteń et al., 1989a; Liao et al., 1991; Hjerteń et al., 1992a; Hjerteń et al., 1993a; Li et al., 1994; Silberring et al., 1994; Mohammad and Hjerteń, 1994; Sedzik et al., 1995; Li et al., 1995; Liao et al., 1996a), hydrophobic-interaction chromatography (Liao et al., 1991; Zeng et al., 1996), reversed-phase chromatography (Hjerteń et al., 1992a; Hjerteń et al., 1993a,b; Liao et al., 1996b; Ericson et al., 1997), normal-phase chromatography (Marusˇka et al., 1999), chiral-recognition chromatography (Hjerteń


Figure 2. Different modes of standard and capillary chromatography on continuous beds. The chromatograms I-IV refer to separation of macromolecules (proteins), and chromatograms V-VII refer to low-molecular-weight compounds. (I) Cation-exchange chromatography on (a) a compressed standard column, i.d. 6 mm [the continuous bed was prepared in a beaker (off-tube synthesis), transferred to a chromatographic tube and compressed]; (b) a noncompressed capillary column, i.d. 0.32 mm. A comparison of the electropherograms in

Ind. Eng. Chem. Res., Vol. 38, No. 4, 1999 1209 a and b shows that there was no loss in resolution with a decrease in bed diameter (Li et al., 1994). (II) Reversed-phase chromatography on a compressed 6-mm i.d. bed at flow rates of (a) 1 and (b) 10 mL/min. Obviously, there was no deterioration in resolution with an increase in flow rate (Hjerteń et al., 1993b). (III) Chromatofocusing of hemoglobins. The bed was synthesized in situ in a 0.32-mm i.d. column by Dr. Yi-Ming Li in the author’s laboratory. For a batchwise, off-tube synthesis, see Hjerteń et al. (1992b). (IV) Hydrophobicinteraction chromatography on columns with inner diameters of (a) 6, (b) 0.32, (c) 0.025, and (d) 0.015 mm. Observe that the resolution was not worsened upon a decrease in bed diameter (Zeng et al., 1996). (V) Chiral-recognition chromatography of practolol, a β blocker, with increasing concentration of the buffer. Cellulase, the enantioselective agent, was immobilized on a continuous bed, prepared batchwise. The i.d. was 6-mm (Mohammad et al., 1993). (VI) Capillary electrochromatography of polyaromatic hydrocarbons. The C18 derivatized continuous bed contained sulfonic acid groups, and the i.d. was 75 µm. (a) Isocratic elution (blank experiment). (b) Gradient elution (the gradient was generated simply by a higher concentration of acetonitrile in the anode vessel than that in the capillary; the sample moved toward the cathode). Evidently, the gradient elution afforded much higher resolution. (c) Experiment performed in the presence of SDS at a concentration below the CM, critical micelle concentration. (d) The same experiment in the absence of SDS. The nonpolar dodecyl groups in this surfactant interacted hydrophobically with C18 groups in the continuous bed, with the sulfate groups probably pointing outward. By this dynamic coating, the operating density of both hydrophobic (C12 + C18) and negative (sulfonic and sulfate) ligands increased, resulting in higher resolution (Liao et al., 1996c). Probably, the dodecyl groups also interacted with the polyaromatic hydrocarbons. (VII) Normal-phase chromatography of phenols and aromatic amines (Marusˇka et al., 1999). Reproduced with permission of the publisher.

et al., 1992a; Mohammad et al., 1993), bioaffinity chromatography (Mohammad and Hjerteń, 1994), dyeligand affinity chromatography (Mohammad et al., 1995), immobilized liposome chromatography (Zhang et al., 1996), chromatofocusing (Hjerteń et al., 1992b), and electrochromatography (Hjerteń et al., 1995; Liao et al., 1996c; Ericson et al., 1997, Hjerteń et al., 1998). The methods for the synthesis of the stationary phases for these modes of chromatography have been described in detail in the references and will not be repeated herein. However, to facilitate for the reader to understand the general discussions about continuous beds, the preparation of one bed (that for electrochromatography) is described in the next section. The principle is the same as that for the preparation of columns for other chromatographic modes. Some illustrative chromatograms are shown in Figure 2. The Preparation of the Continuous Polymer Bed, Exemplified by Synthesis of a Bed for Electrochromatography One-Step Synthesis. This alternative is used when all monomers are water-soluble but is also applicable when one or more of the monomers is nonpolar but can be made soluble in an aqueous medium by addition of a surfactant (Liao et al., 1996c) or a solvent miscible with water but less polar. As an example of a typical one-step in situ synthesis, the preparation of a bed for capillary electrochromatography (CEC) is described. The reasons why CEC is put in focus are that, at present, it is popular enough to be called “the method of the year” and that the synthesis method is similar to those used to prepare beds for other modes of chromatography (the CEC method is outlined in a section below). Because the separations are based on the reversed-phase partition mode, the monomer corresponding to the hydrophobic ligand is not water-soluble. In the description below, this monomer is stearyl methacrylate. However, it dissolves upon addition of Triton X-100, a surfactant. The reaction mixture contains vinyl sulfonic acid, because a charged ligand is required to generate an electroendosmotic flow when a voltage is applied over the column. The following description of the preparation of the bed gives all of the details (Liao et al., 1996c). Piperazine diacrylamide (0.12 g), methacrylamide (0.075 g), vinyl sulfonic acid (150 µL), and ammonium sulfate (0.065 g) were dissolved, in that order, in 1 mL of 0.015 M Tris-HCl (pH 8.5). To 400 µL of this solution (at room temperature) was added 50 mg of stearyl methacrylate. After it was degassed for 2 min with N2,

the solution was supplemented with 15 µL of Triton X-100 (at room temperature). After being heated in a water bath at 65 °C for 5 min and mixed by sonication at room temperature for 2 min, 5 µL of a 10% (w/w) aqueous solution of ammonium persulfate was added. Polymerization was then initiated by adding 4 µL of TEMED. This monomer solution was immediately sucked into the fused silica tubing (coated with [γ-(methacryloxy)propyl]trimethoxysilane), because gel formation started within 1 min. The polymerization proceeded overnight. By means of an HPLC pump and splitting of the solvent, the capillary was flushed with deionized water, acetonitrile, and finally with the mobile phase at maximum pressures of 60, 100, and 150 bar for 100-, 75-, and 25-µm-i.d. columns, respectively. Ammonium sulfate has the important function of inducing such strong hydrophobic interaction among the polymer chains formed that they aggregate into nonporous particles. The void created between the particles is large enough to permit passage of the eluent. A scanning electron micrograph is presented in Figure 3a. Multistep Synthesis. As an alternative to the use of a surfactant to solubilize water-insoluble monomers in a one-step polymerization, a hydrophobic compound (ligand) can be dissolved in an organic solvent and coupled to the hydrophilic matrix in a second step. The coupling can be performed on or off the column. The bed material derivatized by the latter approach can be produced on a large scale for the packing of a great number of columns. A complete description of the synthesis is found, for instance, in Hjerteń et al. (1993b) and Ericson et al., (1997). Continuous-bed-based cation and anion exchangers are commercially available from Bio-Rad Laboratories, Hercules, CA, under the trade name UNO. Packing a Compressible Column The column tube (an appropriate design is described in a paper by Hjerteń, 1991a) is filled with the bed material (for the preparation of beds for different modes of chromatography, see “Chromatograms Illustrating the Multitude of Chromatographic Modes Continuous Beds Offer for both Standard and Capillary Chromatography”). A piston is inserted and moved down to compress the bed. An HPLC pump is attached and operated at a flow rate somewhat higher than that to be employed in the subsequent chromatographic experiment. The piston is pressed down to make contact with the bed if a void forms at the top of the column. Upon compression of the bed, the interconnecting channels


1 and 2 can be solved by using continuous beds, because (independently of the column diameter) they are prepared by sucking a monomer solution into the chromatographic tube (a piece of fused silica tubing) followed by polymerization, and no frit is required because the bed becomes attached covalently to the tubing wall. Problem 3 can be completely overcome by employing a recently introduced pump based on thermal expansion of a liquid (Ericson and Hjerteń, 1998) and problem 4 by creating a smooth gradient in a capillary by filling it stepwise with sections of the eluent of different concentrations (Li et al., 1994). The chromatograms in Figure 2 (b in part I, part III, b-d of part IV, and part VII) refer to capillary chromatographic experiments. Chromatography in narrow-bore tubes has many names; capillary chromatography, microchromatography, narrow-bore tube chromatography, and nanochromatography. The method is the chromatographic counterpart of capillary electrophoresis. I prefer the first name, simply because analogous methods should be given analogous names (Hjerteń et al., 1998a). Capillary Electrochromatography (CEC) Figure 3. (a) Scanning-electron micrograph of a nonpacked continuous polymer support, prepared batchwise. The white bars represent a length of 10 µm. If the photo gives a true picture of the bed, it is composed of walls of aggregated particles and channels between the aggregates through which the eluent can pass (Liao et al., 1991). (b) Photomicrograph of a continuous polymer bed, synthesized in situ in a capillary with an i.d. of 10 µm (Li et al., 1994). Reproduced with permission of the publisher.

become more narrow, which increases the resolution considerably, as discussed in “To What Extent Can the Zone Broadening in a Chromatographic Gel Bed Approach the Extremely Small Broadening Characteristic of Gel Electrophoresis?” This is an approach which can be applied on many types of beds (Fishman and Barford, 1970; Edwards and Helft, 1970) but is of practical importance only if the bed is designed such that the flow rate does not decrease significantly upon compression. The continuous beds [and the agarose beds (Liao and Hjerteń, 1988; Hjerteń et al., 1991b)] have the unique property that they have low flow resistance even following compression. The importance of compression to increase the resolution is illustrated in Figure 1. Capillary Chromatography on Continuous Beds Capillary chromatography has not become as popular as is its electrophoretic counterpart, capillary electrophoresis. There are several reasons for this, the following four being crucial: (1) the difficulty of packing a narrow-bore column uniformly with small beads is significant, (2) the frits required to support a packed bed increase the zone broadening and may be difficult to prepare and install (in capillary electrochromatography, the frits generate air bubbles, which spoil any experiment), (3) commercial pumps which permit pulsefree flows at flow rates in the nL/min range are not commercially available, and (4) the difficulty to create extremely small-volume gradients is obvious. The latter two problems can be partially overcome by using standard HPLC pumps in the splitting mode. However, in doing so, one of the advantages of capillary chromatography, the low consumption of organic solvents, is lost. In addition, the actual flow through the column can vary and is not easy to determine. Problems

Zone broadening in chromatography can be reduced by decreasing the diameter of the beads (the average diameter of the channels in a bed). However, in practice, there is a limit in particle size which one cannot go below in pressure-driven chromatography because of the resulting high back pressure. This limit can be reduced considerably if the flow through the column is achieved by electroendosmotic instead of hydrodynamic flow. A mechanical pump is thus not required. However, we cannot approach particle sizes of the same dimensions as those of the pores in polyacrylamide gels, because the electroendosmotic mobility under such conditions is low, owing to overlap of the Helmholtz double layers (Knox and Grant, 1987; 1991). Some theoretical and practical difficulties must be overcome before the elegant method of CEC can become generally applicable. The technique, along with electrophoresis/chromatography in solutions of charged polymers and charged gels, permits analyses of noncharged substances, which conventional electrophoresis does not allow. These methods are used most often in the reversed-phase chromatography mode (see Figure 2VI). The fact that continuous beds do not require a supporting frit and can easily be synthesized in very narrow capillaries makes them appropriate for capillary electrochromatography (see previous section). Capillaries with diameters as small as 25 µm are preferable from the point of view that the run times become short because the thermal zone deformation in such narrow tubes often is negligible even at high field strengths. Continuous beds are likely to be chromatographically more homogeneous than are packed beds, to judge from the observation that the plate height in pressure-driven chromatography on continuous beds (which consist of nonporous particles) is close to that obtained in CEC (Figure 4). For beds packed with macroporous beads, the difference is larger (Knox and Grant, 1991; van den Bosch et al., 1996). An explanation could be that the electroendosmotic flow in the pores transports the analytes into and out of a bead faster than does diffusion. For gradient elution of proteins in CEC in the reversed-phase mode, see Ericson and Hjerteń (1999).


Figure 4. Dependence of plate height on linear velocity for electroendosmosis- and pressure-driven flow. The difference in plate height is very small, indicating that the continuous beds are chromatographically very homogeneous (Ericson et al. 1997). Reproduced with permission of the publisher.

nonlaminar flow (perhaps local rotations of the mobile phase) which moves the solute more rapidly from one bead (channel wall) to another than does diffusion (faster mass transfer). The gel particles of the continuous beds are made nonporous to accelerate the mass transfer. As nonlaminar flow sets in, its magnitude can be expected to increase with an increase in flow rate. Accordingly, the higher the flow rate, the narrower the peaks should be, as is experimentally found (Figure 5). It should be stressed that we have no experimental evidence that this hypothesis based on a nonproven nonlaminar flow is correct. The experimental conditions for the preparation of continuous polymer beds [and the nonporous beads constituting the agarose columns (Liao and Hjerteń, 1988; Hjerteń et al., 1991a,b)] have been chosen such that the channels (beads) have a rough surface, which should induce nonlaminar flow at a lower flow rate than do smooth surfaces. The rough surface also increases the surface area considerably which, along with the small bead size, explains why the binding capacity for proteins is about the same as that of smoother macroporous beads (Mohammad and Hjerteń, 1994). The above attractive resolution-flow rate relationship refers to gradient elution experiments with proteins, which only exceptionally, i.e., at very low ligand densities (Yao and Hjerteń, 1987), can be eluted isocratically. However, we have found the same relationship for low-molecular-weight compounds upon isocratic elution in normal-phase chromatography (Marusˇka, personal communication). Bead Diameter and Flow Resistance in a Virtual Packed Column, Equivalent to a Continuous Bed The flow resistance, L, of a packed bed can be calculated from the equation

u)

Figure 5. Plot of resolution of ribonuclease and cytochrome C against flow rate in reversed-phase chromatography on a compressed 6-mm i.d. bed (Hjerteń et al., 1993b). Reproduced with permission of the publisher.

The Attractive Relationship between Resolution and Flow Rate for Continuous Beds Typical of these beds is that the resolution often is independent of the flow rate. For compressed beds, the resolution can even increase with an increase in flow rate (Figure 5). Compression of beds in capillary columns should be avoided as it cannot easily be done uniformly because voids often form in the bed. As an alternative to compression of the bed, the gel concentration should be higher in capillary continuous beds, which makes them more rigid [less compressible (Liao et al., 1996a-c)]. The increase in resolution with an increase in flow rate may be due to the generation of

Pd2 LηL

(1)

where u ) the linear flow rate, P ) the pressure drop, d ) the average diameter of a bead, η ) the viscosity, and L ) the bed height (Karger et al., 1973; Snyder and Kirkland, 1979). The Diameter of the Beads in an Equivalent Packed Bed. Consider a packed bed with flow properties equivalent to those of a continuous bed; i.e., the two beds have the same flow resistance, L, and therefore they afford the same flow rate (u) when the experiments are performed in the same buffer (η is the same) at the same pressure (P) on beds of the same height (L). The analysis times are, accordingly, the same. The small particles making up a continuous bed (Figure 3a) are nonporous, and therefore we also assume that the beads in the equivalent packed bed are nonporous, i.e., L ) 250 (Snyder and Kirkland, 1979). To estimate the diameter of these beads (dequiv) according to eq 1, we take data from the experiments presented in Figure 5 in Hjerteń et al. (1993a) and employ CGS units (u ) 1.3 cm s-1, P ) 12 bar ) 12 × 106 dynes cm-2, L ) 250, η ) 0.009 P (0.1% TFA in acetonitrile), L ) 11.7 cm). The approximate value of dequiv can then be calculated at 15 µm. A packed bed with nonporous beads of a diameter of 15 µm has, consequently, the same flow properties as the real continuous bed. It should be stressed that the equivalent diameter and the equivalent flow resistance discussed in the next section only


refer to flow properties of the equivalent packed bed. No conclusions can be drawn as to other characteristics of the virtual equivalent bed or the real continuous bed. The Flow Resistance of an Equivalent Packed Bed. By reference to the experiment above and by the assumption that the average diameter of the gel particles in the continuous bed is around 0.5 µm (see the scanning electron micrograph in Figure 3a), eq 1 gives Lequiv ) 3, i.e., the flow resistance of the equivalent packed bed is considerably lower than that of a regular packed bed [pellicular (nonporous) packings, L ) 250; spherical porous packings, L ) 500; irregular porous packings, L ) 1000]. An explanation of the extremely low flow resistance could be that the tiny gel particles making up a continuous bed are connected by covalent bonds and thus form a rod. The large uncertainty in the estimation of the effective diameter, d, of the gel particles in the continuous bed makes the calculated equivalent L-value of 3 extremely approximate, particularly since d is squared in eq 1. However, for the qualitative discussion above, it is only of interest that Lequiv , 250. In other words, the equivalent packed bed must have a very low L to permit the same flow rate as the continuous bed (1.3 cm s-1) at the pressure applied (12 bar). The experiments from which the data are taken were performed on a 6-mm column. Capillary columns give still lower flow resistance, since the bed is attached to the capillary wall and, therefore, cannot be compressed. For the same reason, no frits, which often clog the bed and create a high flow resistance, are required. The influence of the composition of the bed on the flow resistance has been studied by Nakazato et al. (1994). Completely Homogeneous Continuous Gel Beds The continuous beds described above are probably chromatographically more homogeneous than conventional packed beds, but the zone broadening caused by Eddy diffusion is not negligible, because they consist of particles (although very small). Gels, for instance, of agarose and polyacrylamide, should be the ideal chromatographic media because they are not made up of any particles. They are, thus, physically completely homogeneous. However, the pores in a gel are too small to permit a transport of the mobile phase by a hydrodynamic flow. The problem can be overcome if this flow is replaced by an electroendosmotic flow. The method was suggested as early as 1950 for molecular-sieving of neutral hydrolysis products of amylose (Synge and Tiselius, 1950). Although this is an obvious approach, it has not been used since then. The probable reasons are (1) that the above experiment was not conclusive and (2) the great difficulties encountered in designing gels with the properties that theoretically are required for high electroendosmotic flow. For instance, because of sterical hindrance, agarose chains cannot come sufficiently close to each other to form a gel (via hydrogen bonds) following attachment of ligands for interaction with the analytes and for generation of a high electroendosmotic flow. This and other problems have been overcome (Hjerteń et al., 1998b). The experiment presented in Figure 6 is an example of both a separation in a homogeneous continuous bed and a simple method for preparation of a gel with the desired chromatographic properties. The sample consisted of a mixture of nonionic aromatic compounds. The capillary was filled with a dilute buffer containing poly(vinyl alcohol) and the electrode vessels with a borate buffer. When a

Figure 6. Electrochromatography in a completely homogeneous gel. Sample: methyl, ethyl, propyl, and butyl esters of p-hydroxybenzoic acid. Gel: 4% poly(vinyl alcohol), cross-linked with borate ions. Capillary: noncoated, 25-µm i.d. × 25 cm. Voltage: 6000 V. Reproduced with permission of the publisher.

voltage was applied, the borate ions migrated into the capillary and formed a charged complex with the polymer. The borate ions had the dual function of transforming the polymer solution to a gel by crosslinking and generating electroendosmosis. The separations are based on the so-called aromatic adsorption of the analytes to the polymer chains (Gelotte, 1960). The method has the great advantage that the synthesis involving attachment of ligands can be omitted. The homogeneous gels can with some justification be considered the second generation of the continuous beds because they have all of the attractive properties of the earlier ones and, in addition, are perfectly homogeneous and have much narrower flow channels (often in the range of 50-1000A); i.e., the potential resolution should be higher. For more information about this new type of beds including the preparation of other gels and theoretical aspects, see Hjerteń et al. (1998b). Conclusions Continuous chromatographic beds have many important characteristic features: they are prepared by a simple polymerization procedure in situ in the chromatographic tube (or, if so desired, batchwise); they can be synthesized in columns of any diameter and are, therefore, preferable to packed beds for capillary chro-


matography, including electrochromatography (the novel thermal micropump should contribute to making capillary chromatography more popular); contrary to classical chromatographic theory, their resolution is often independent of flow rate or may even increase upon an increase in flow rate; and their back pressure is relatively low. The Eddy diffusion is negligible for the recently introduced completely homogeneous continuous gel beds. Acknowledgment The continuous bed project has been supported economically by the Swedish Natural Science Research Council, the Swedish Research Council for Engineering Sciences, the Carl Trygger Foundation, and the Swedish Institute. Literature Cited van den Bosch, S. E.; Heemstra, S.; Kraak, J. C.; Poppe, H. Experiences with Packed Capillary Electrochromatography at Ambient Pressure. J. Chromatogr., A 1996, 75, 165. Edwards, V. H.; Helft, J. M. Gel Chromatography: Improved Resolution through Compressed Beds. J. Chromatogr. 1970, 47, 490. Ericson, C.; Liao, J.-L.; Nakazato, K.; Hjerteń, S. Preparation of Continuous Beds for Electrochromatography and ReversedPhase Liquid Chromatography of Low-Molecular-Mass Compounds. J. Chromatogr. A. 1997, 767, 33. Ericson, C.; Hjerteń, S. Pump Based on Thermal Expansion of a Liquid for Delivery of a Pulse-Free Flow, Particularly for Capillary Chromatography and Other Micro-Volume Applications. Anal. Chem. 1998, 366, 6-372. Ericson, C.; Hjerteń, S. Reversed-Phase Electrochromatography of Proteins, Using Normal Flow and Counter flow Gradients. Anal. Chem. 1999, in press. Fields, S. M. Silica Xerogel as a Continuous Column Support for High-Performance Liquid Chromatography. Anal. Chem. 1996, 68, 2709. Fishman, M. L.; Barford, R. A. Increased Resolution of Polymers through Longitudinal Compression of Agarose Gel Columns. J. Chromatogr. 1970, 52, 494. Gelotte, B. Studies on Gel Filtration Sorption Properties of the Bed Material Sephadex. J. Chromatogr. 1960, 3, 330. Hileman, F. D.; Sievers, R. E.; Hess, G. G.; Ross, W. D. In Situ Preparation and Evaluation of Open Bore Polyurethane Chromatographic Columns. Anal. Chem. 1973, 45, 1126. Hjerteń, S. Free Zone Electrophoresis. Chromatogr. Rev. 1967, 9, 122-219. Hjerteń, S. Dedication to Professor Arne Tiselius. Ann. N.Y. Acad. Sci. 1973, 209, 5. Hjerteń, S. The History of the Development of Electrophoresis in Uppsala. Electrophoresis 1988a, 9, 3. Hjerteń, S.; Kunquan, Y.; Liao, J.-L. The Design of Agarose Beds for High-Performance Hydrophobic-Interaction Chromatography and Ion-Exchange Chromatography which Show Increasing Resolution with Increasing Flow-Rate. Makromol. Chem. Symp. 1988b, 17, 349. Hjerteń, S.; Liao, J.-L.; Zhang, R. High-Performance Liquid Chromatography on Continuous Polymer Beds. 1989a, 473, 273. Hjerteń, S.; Valtcheva, L.; Elenbring, K.; Eaker, D. HighPerformance Electrophoresis of Acidic and Basic Low-MolecularWeight Compounds and of Proteins in the Presence of Polymers and Neutral Surfactants. J. Liq. Chromatogr. 1989b, 12 (13), 2471. Hjerteń, S. High-Performance Agarose-Based Chromatographic Media and Their Applications in Biopolymer Separation, in M. T. W. Hearn (Ed.), HPLC of Proteins, Peptides and Polynucleotides. Contemporary Topics and Applications, VCH Publishers: New York 1991a, pp 119-148. Hjerteń, S.; Mohammad, J.; Eriksson, K.-O.; Liao, J.-L. General Methods to Render Macroporous Stationary Phases Nonporous and Deformable, Exemplified with Agarose and Silica Beads and Their Use in High-Performance Ion-Exchange and Hydrophobic-

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Received for review September 22, 1997 Revised manuscript received July 27, 1998 Accepted August 17, 1998 IE970676B

Standard and Capillary Chromatography, Including

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