A Critical Appraisal of Capillary Electrochromatography - Analytical

ical Appraisal

hough still in its infancy, CEC

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ometimes the whole is greater than the sum of its parts. Analytical chemists see this when several techniques are combined to create a new technology. The goal is to incorporate the most favorable properties of various technologies into a single, more powerful system. This basic concept is exploited in capillary electrochromatography (CEC), which aims at combining the best aspects of CE and capillary (cap) HPLC. However, new technologies can bring not only benefits but also raise unsolved questions and technical problems. Such is the case with CEC. Does CEC generate a higher number of theoretical plates than cap-HPLC (because of the pluglike profile) when capillaries of the same dimensions and the same stationary phase are used? Can small micrometer-sized packing materials (0.5–2.0 µm) be used in CEC when they would not be an option in cap-HPLC because of high backpressure? Is the retention of analytes simply the sum of electrophoretic migration and chromatographic retention terms, or should the physical description of the migration processes in CEC include considerations about a variety of simultaneously occurring independent and dependent processes? What is the advantage of CEC for practical applications compared with CE and capHPLC? In this article, these and other questions central to the emerging applications of CEC as an analytical procedure for bioanalysis are considered. In particular, the potential of this orthogonal technique for the nanoscale separation of peptides and proteins is appraised.

Basics CEC is a capillary-based analytical technique that typically uses fused silica capillaries; other options based on microfabrication and different classes of polymeric capillary materials can be contemplated. CEC can be operated either in the packed mode (pCEC) with microparticulate stationary phases and monolithic continuous-bed sorbents or in the etched open-tubular format (ot-CEC). However, the most widely used are microparticulate sorbents similar to those used in reversed-phase cap-HPLC. Just as in cap-HPLC, the eluents used in CEC are typically mixtures of organic solvents and aqueous buffers. Electroosmosis transports the mobile phase and the analytes through the capillary. The migrational velocities of the analytes are controlled in part by the magnitude of the electroosmotic flow (EOF), which is generated at solid–liquid interfaces in packed or etched ot capillaries when an electrical field is applied across both ends of the capillary. The bulk migration of the mobile phase, which depends on the magnitude of the electrical field, exhibits a characteristic pluglike flow profile directed toward the cathode in silica-based systems. The phenomenon of electroosmosis is thus a direct consequence of the chemical characteristics of the fused silica capillary wall and the packed particles themselves— both of which are in contact with the background electrolyte. Electroosmosis is a well-described phenomenon that has been used in numerous techniques. The first experiments exploiting EOF in CEC date back to Pretorius’s work in 1974, but this pioneering study attracted very little attention for some

A P R I L 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y

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These events include electrostatic, atyears (1). However, pivotal observatractive, and repulsive interfacial forces, tions made in 1981 by Jorgensen and the magnitude of which depends on Lukas (2) and later extended in the the propensities of the chromatomid-1980s by Tsuda (3), Knox (4), graphic sorbent to undergo selective and others paved the way for CEC to types of interactions. The result is that emerge as a powerful technique for the within this conceptual framework, separation of neutral analytes. As a conCEC provides very high selectivity, sequence, the number of CEC appliwhich is particularly useful for resolvcations for low-molecular-weight noning charged compounds. Moreover, polar and polar organic molecules has compared with ot-CE, greater control increased significantly and has been µEOF over selectivity can generally be documented in several recent reviews achieved for polar compounds because (5, 6). Yet, as an analytical method, of surface–solute interactions, and for particularly for the separation of bioµcations nonpolar analytes because of associmolecules, CEC appears to be far from ated sorption and partitioning effects, becoming a routine technique. when these analytes are subjected to To describe separation performance µanions the CEC separation modes. Finally, in p- or ot-CEC, the height equivalent exotic mobile-phase additives, such as of a theoretical plate (HETP) can be detergents, ion-pairing reagents, and used, just as with cap-HPLC, but the crown ethers, can be used to enhance concept must be adapted to incorpoElectrochromatographic retention kcec selectivity with CEC, just as with rate several other contributions. In ot-CE. cap-HPLC, the dominating term conIn addition to the dependence on tributing to the plate height for an unsurface-mediated contributions, the FIGURE 1.Schematic of the different separation retained compound migrating at its net retention of a charged analyte is mechanisms operating in CEC resulting from optimum linear velocity is the eddy affected by the direction and magnithe charge-to-size ratio of the bioanalyte sudiffusion term A (7 ). It is generally actude of the electrokinetic velocity vecperimposed on its chromatographic retention. cepted that the minimal HETP that tor ␷e, which arises due to the electrocan be achieved in cap-HPLC with phoretic mobility relative to the EOF capillaries well packed with particles of the same uniform diameter is approximately two times the mobility µeof. This situation is exemplified in Figure 1 for neuparticle diameters. Because of the pluglike flow profile in CEC, tral, anionic, and cationic analytes. The EOF is directed toward the HETP is expected to be smaller, and the velocity of the EOF the cathode for negatively charged sorbents, with the velocity vecis a very weak function (i.e., almost independent) of the particles’ tor in the same direction for cations and in the opposite direction diameters. Moreover, the film mass transfer coefficient is ap- for anions. However, superimposed on the electrophoretic migraproximately proportional to the average diameter of the pack- tion is the prevailing chromatographic effect. By using an unretained substance as the EOF marker, it is ing material in p-CEC or, in the case of microchannel devices, varies with the size of the channel (8, 9). Thus, for electroosmot- possible to readily identify those analytes that elute before or after ically driven flows in packed-bed or porous monolithic CEC sys- the marker. Depending on the properties of the sorbent and elutems, the film mass transfer coefficient and the intraparticle EOF ent, positively and negatively charged analytes can elute before or after the EOF marker, depending on the nature of their charge and varies with the size of the interstitial channels for bulk flow. In addition, when electrolytes and buffers are used, intraparti- potential for interacting with the sorbent. Consequently, using cle EOF can significantly contribute to separation efficiency and global charge features to formally differentiate the migrational beresolution by decreasing intraparticle mass transfer resistance and haviors of anionic, neutral, and cationic analytes into specific redispersive mass transfer effects, while increasing intraparticle mass gions of the CEC “separation space” lacks any scientific foundatransfer rates. Therefore, the use of micrometer- and submicrom- tion. Instead, the overall elution time of an analyte in p- or ot-CEC eter-sized particles or monolithic sorbents with interconnecting is the result of the summed contributions from multiple migramacroporous networks can lead to even smaller values of HETP tional effects. Thus, for charged analytes, the correct questions are, and much higher plate numbers in CEC than in classical cap- “What is the magnitude of the electrophoretic contribution?” HPLC. Moreover, CEC does not pay the penalty of high back- and “What is the consequence of the sorption/partitioning conpressures with such packing materials that is seen with cap-HPLC. tribution on the total retention?” One of the molecular attributes that dictates the nature of the To answer these questions, the theoretical foundations of mechanisms of separating charged analytes in CEC is the differ- CEC must be examined in terms of the structure of condensed ence in their electrophoretic mobilities. Superimposed on this matter. Moreover, the theoretical foundations of CEC need to process are chromatographic retention events based on the dif- be validated with real samples, in which secondary effects can ferent physical and chemical characteristics of the analytes. cause the behavior of the sample to diverge from the theoreti-

cal ideal. It has often been tacitly assumed that the chromatographic retention of all analytes on n-alkyl bonded silica sorbents in p- or ot-CE is equal or similar to that observed with reversedphase cap-HPLC. This might be true for nonpolar compounds; however, with polar or ionic analytes, the sorption/partitioning effects will probably also be significantly affected by the electrical field strength and the double layer properties at the surfaces of the capillary wall and/or the surface of the particles. The result is that the kinetic and thermodynamic processes involved in the retention behavior of polar analytes, such as peptides and other biomolecules, in p- or ot-CEC are much more complex than in CE and cap-HPLC, respectively.

Instrumentation

In CEC, hydrodynamic (by regulating pressure) and electrokinetic (by applying a voltage) injections are used. Although electrokinetic injection is the most commonly used method, its primary problem is that molecules with different mobilities (due to differences in their charge-to-size ratios) show pronounced dependencies on their injection concentrations. For example, it may be impossible to inject an appropriate amount of some anionic-charged compounds onto silica-based systems because these analytes move in the direction opposite to the EOF. Reproducible hydrodynamic injection is not practical because of inexact control over the higher pressures necessary for packed CEC capillaries (>2 bar). In the first step of the injection procedure, the sample is injected as a plug and is then followed by the background electrolyte. The permeability through a p-CEC capillary varies because the capillary consists of frits and packed and open sections, which leads to different EOF velocities and possible cavity formation. To avoid cavities, the inlet and outlet ends are usually pressurized to 10 bar. One of the more critical issues in p- or ot-CEC is the batch-tobatch reproducibility of packed and chemically modified, etched ot capillaries (11–13). Although numerous p-CEC capillaries are now commercially available, capillary and frit reproducibility remains a challenge. (A table of vendors can be found in Supporting Info at pubs.acs.org/ac.) Nevertheless, p-CEC capillary lifetimes of >1000 consecutive analyses have been reported (11). Typical capillary packing materials include n-octadecyland n-octyl-bonded silicas of ~3-µm average particle size or chemically modified silicas with strong cation or anion functionalities. A growing interest in mixed-mode phases, containing both n-alkyl ligands and strong cation exchange groups (sulfonic acids), has arisen because a high EOF can be obtained over the larger pH interval of 2.0 to 8.0. The overall charge density responsible for the magnitude and direction of the EOF is more difficult to estimate when strong anion exchange groups, such as quaternary ammonium derivatives, are attached to silica-supported materials. Thus, if the pH

The same instrumentation is used in CEC as CE but with some additional components. For “pure” CEC, no external pressure is applied at the inlet side of the capillary. In pressurized CEC— also known as electrically assisted cap-HPLC—a pressure differential of >40 bar is applied across the capillary. A schematic of a CEC instrument interfaced to UV and MS detection via a sheath liquid interface is shown in Figure 2. Polyimide-coated fused silica capillaries with 50- to 100-µm i.d.s and 80- to 600-mm lengths are typically used, although capillaries up to a 1000-µm i.d. have been tried. In principle, three types of CEC capillary systems exist— microparticulate-packed, ot, and continuous-bed (monolithic) (10). Most commercial CEC capillaries are packed with 3-µm silica-based particles. When operational, the ends of the capillary dip into two buffer vials, with electrodes connected to a power supply generating up to 30 kV mounted in the vials. A section of the polyimide coating is removed from both ends of the capillary and immediately after the separation zone to enable UV detection. Figure 2b shows a schematic for a coupled CEC/electrospray ionization (ESI) mass spectrometer using a sheath liquid interface. The inlet side of the capillary is immersed in the anode’s buffer vial together with the separation electrode. The other end of the capillary is inserted through a membrane into the stainless (a) (b) steel needle of the sheath liquid interface High-voltage supply High-voltage supply through which the electrospray voltage is applied. The apparent separation voltage is + – + – the applied inlet voltage minus the ESI voltage. The outlet end of the packed capMass Capillary Capillary illary is blunt-cut immediately after the spectrometer outlet frit—the capillary is not extended as it is with UV detection. MS/MS adds a dimension to p- or ot-CEC separations in Detector terms of an m/z-sensitive detector that Stainless Sheath makes it possible to acquire detailed inforsteel liquid mation about the structure of the analyte. needle Buffer vial For example, the limits of peptide detecBuffer vials ESI, high voltage tion by CEC can be greatly enhanced, thus allowing the analyte concentration to be decreased by ~200-fold for MS deFIGURE 2.Schematic for CEC instrumentation (a) with UV detection and (b) coupled to an tection compared with UV detection. electrospray ionization mass spectrometer.

of the mobile phase is 2.0, the net charge of the sorbent depends on contributions from both the ionized silanol groups and the quaternary ammonium groups. Instead of using chemically bonded silica packing materials, the desired ligands can also be directly attached to the surface of the capillary wall (etched ot capillaries). The capillary wall is usually etched to increase the surface area by a factor of 1000, which increases the ligand density in ot-CEC and thereby the extent of solute–surface interactions (14). Most CEC analyses have been performed with n-octadecyl-, noctyl-, or mixed-mode-bond porous microparticulate silicas. However, based on the initial work of Hjertén, various research groups have successfully developed synthetic procedures for the in situ preparation of monolithic polymers, including polymethacrylates, polyacrylamides, and polystyrenes, as continuous beds in CEC capillaries (15–18). Monolithic silica capillaries have also been developed for p-CEC (19), and these columns are attracting increasing attention because they eliminate some of the problems associated with frit formation and minimize the effects of fused silica capillary deformation that can arise when spherical, porous silica-based particles are poorly packed. The overall goal with monolithic CEC systems is to develop robust columns, and recent studies provide encouraging data that these columns do last and perform well. Nevertheless, CEC capillaries packed with bare silica parti-

toluene, and a photoinitiator to UV light at 365 nm for 5 min (23). Analogous procedures can be used to prepare sol–gel monolithic columns with reversed-phase behavior for analytical and semipreparative applications (24, 25). Finally, the role of the frit or the capillary wall in nonspecific adsorption events or system nonreproducibilities should not be overlooked. Laser-induced fluorescence imaging based on a XeCl excimer laser coupled to an image-intensified charge-coupled device camera is an effective technique for collecting information on separation dynamics along the capillary column or at the frit (26). The dynamics are followed by continually updating fluorescence intensity profiles on a computer display.

Operation of CEC capillaries Before packed or surface-modified ot capillaries are used, they have to be conditioned. This can take place either in the instrument or externally by using a conventional HPLC pump. The first step is to control the EOF of the capillary as a function of the field strength E (V/cm), which is the ratio of the applied potential V and the total capillary length L. In the case of p-CEC, L is the sum of the packed and the ot sections on either side of the frits, assuming that UV detection is used. The EOF increases linearly with increasing potential assuming that negligible joule heating or cavity formation occurs. The maximum EOF that can be achieved with a 50-µm i.d. capillary packed with porous 3-µm particles and operated with an electric field strength of ~800 V/cm is ~3 mm/s. To generate these

One of the more critical issues is batch-to-batch reproducibility of cles can be efficient and selective for analyzing many basic types of analytes (20). However, when it comes to analyzing peptides and proteins, reversed-phase or mixed-mode sorbent systems are the preferred options. CEC capillaries can be packed with microparticulate packing materials either by the well-known slurry technique or electrokinetically by applying a high electrical field (10, 11). The crucial step is generating frits. Normally, 2-mm-thick porous frits are created during the heating procedure by sintering the silicabased sorbent under hydrothermal conditions. One way to avoid frits and thereby diminish cavity formation in p-CEC is to utilize the keystone effect, in which the i.d.s of capillary end-pieces are 10-fold smaller than the average particle diameter (21). Particulate silica-based sorbents can also be used to manufacture continuous beds by hydrothermal treatment of the whole packed capillary, forming a stable agglomerate. In this case, the permeability of the bed is enhanced during operation under optimal EOF velocity conditions due to the absence of frits (22). An alternative approach is to generate porous frits in p-CEC by exposing a plug of sol–gel in the presence of HCl, water,

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EOF velocities, optimum conditions are required, such as a highpH background electrolyte and a high concentration of accessible silanol groups on the stationary-phase surface. However, the EOF is affected by other parameters, including the chemical composition of the sorbent; the type of ligand at the surface of the packing material; the charge density at the sorbent surface; capillary temperature; and the eluent properties, such as pH, ionic strength, content of organic solvent, and buffer type. At very low pH, the EOF is usually low with reversed-phase silica-based sorbents in p-CEC because of the partial protonation of the ionized silanol groups. The isoelectric point of silica is approached at pH 2.0–3.0. However, with chemically modified silicas containing strong cation or anion functionalities, the EOF is nearly independent of the pH in the range 2.0–8.0 (27 ).

Capillary efficiency Analytes are transported along the capillary by the EOF in the interstitial channels and by their intrinsic electrophoretic mobility, which may be in the same or the opposite direction as the EOF depending on the analyte’s charge. This combined mi-

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gration results in axial dispersion in the interstitial channels; film mass transfer and diffusion into the pores when a porous sorbent is used; and intraparticle flow due to the EOF within the stationary-phase pores (21, 28). Currently, the HETP concept as used in cap-HPLC is applied to CEC with minor modifications. Experimental data indicate that excellent capillary performance can be achieved for uncharged analytes on a 3-µm reversed-phase silica column operated at an optimal linear velocity of 1 mm/s with ~200,000 theoretical plates/m (29). Given that lower efficiencies are anticipated because of adsorption and wall effects, the background electrolyte composition becomes more critical when analyzing peptides and proteins. Control of the overall zeta potential ␨total of the system thus becomes a critical consideration for the separation of these biomolecules by p- and ot-CEC. The particle size should be ~2 µm to achieve a minimum plate height under optimal linear velocity conditions with commercial CEC equipment. Capillaries packed with micrometer and submicrometer particles are predicted to require either E > 1000 V/cm or L < 100 mm to operate optimally. When only UV detection is available, it is likely that overload conditions prevail for most analytes separated on surface-modified packed or ot capillaries—that is, the conditions may be near or at the saturation value of the sorption isotherm (as determined from a plot of adsorbed amount vs analyte concentration). Due to constraints on the detection limits with the current generation of UV-based detectors, it has yet to be shown whether CEC capillaries can generally be operated under linear (nonoverload) experimental conditions or if sample loadings at the subattomolar level are always required.

Retention and selectivity For evaluating retention behavior in p- and ot-CEC, analytes can be conveniently grouped into nonpolar, polar, and charged species. Because resolution and selectivity for nonpolar analytes are satisfactory with other separation techniques, they are not a major target for CEC. With the demands of proteomic and zeomic (integrated biopathway systems analysis) studies, attention is shifting to polar and charged analytes, particularly biopolymers such as peptides and polypeptides. Exploiting both selective surface interactions and electrokinetic effects offers some separation attributes that are not available with cap-HPLC or CE (30). Thus, experimentally, it is possible to make a smooth transition from cap-HPLC to p-CEC by adjusting the contribution from pressure-driven and/or electrically driven flow. This adjustment of flow characteristics could result in an infinite number of hybrid techniques, from pure cap-HPLC (no electric field component) to pure p-CEC (no pressure component). By contrast, the transition region between CE and p-CEC is not as continuous. Instead, a conceptual and practical linkage is provided with ot-CEC, although there is not, currently, the same variety of sorbent surfaces available as that found for cap-HPLC and p-CEC systems. Over the past two years, considerable effort has been invested in studying the retention behavior of basic (31–33) and acidic (34) bioanalytes with n-alkyl silicas. In addition, bare silicas appear to

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Time (min) FIGURE 3.Separation of five small peptides on a CEC ODS C8 capillary column. Column, 100-µm i.d., 25-cm separation length, 33.5-cm total capillary length; voltage, 25 kV; background electrolyte, 40 mmol/L NH4Ac–AcOH (ph 4.5):acetonitrile (1:1 v/v); temperature, 20 ºC; detection, 214 nm. T, triptorelin; D, desmopressin; O, oxytocin; A, peptide A; U, uracil (EOF marker); C, carbetocin (uncharged).

have some separation potential for biomolecules by CEC (35). This observation is in accordance with predictions of Liapis and Grimes that a strong affinity between ligands and analytes is not mandatory because many other physicochemical factors contribute to retention and selectivity (36). To adjust the separation parameters accordingly is the key to success. For example, two similar peptides, [Met5]enkephalin and [Leu5]enkephalin, which differ by only one amino acid residue, can be separated on n-octadecyl-bonded silica by exploiting the difference in their hydrophobicities (32). Although the CEC retention of simple solutes can be predicted using cap-HPLC and CE data, the situation becomes more complex with peptides (37–40). Small peptides normally have no well-developed secondary structure and instead exist in random coil conformations in bulk solutions. Because of their size and increasing complexity, polypeptides and proteins adopt preferred secondary and tertiary structures. This results in more complex CEC retention behavior because contributions from small molecular contact regions have more influence than the amino acid sequence of the polypeptide or protein as a whole under the specified conditions of the investigations (32, 33). Peptides and proteins are now generally separated by conventional reversed-phase LC systems under gradient conditions with water–organic solvent mixtures at low pH and ionic strength, or by CE, often with buffers of relatively low ionic strength and pH. The selection of sorbents for reversed-phase LC is large, and the separation mechanisms for peptides and proteins on these columns have been extensively investigated (37 ). Peptides and proteins in


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CE are frequently separated using coated capillaries that diminish interactions from basic side chains with the free silanol groups on the capillary surface, i.e., to suppress EOF. In the other case, the EOF velocity strongly depends on the eluent’s pH, displaying a high value at pH 8.0 and a very low one at pH 2.0. One way to overcome the EOF pH dependency is to co-immobilize strong cation exchange (sulfonic acid) groups with reversedphase ligands to form mixed-mode phases (32, 33). However, depending on the response of the amino acid side chains to the electrolyte pH, polypeptides will exhibit an intrinsic net charge that can be anisotropic in its distribution (41). Thus, selected properties of the eluent will either enhance or suppress the electrostatic interactions between the sorbent and polypeptides in p-CEC—a feature that clearly distinguishes CEC from reversed-phase LC. Thus, it is possible, in specific cases, to use reversed-phase sorbents and CEC to obtain high-resolution separations that cannot be achieved by reversed-phase LC, simply because CEC offers a greater palette of adjustable parameters. The trade-off, of course, is that the optimization of the CEC separation may be more time-consuming and complex.

trophoretic mobilities (Figure 3). On the other hand, if a low salt concentration and a high content of organic solvent are used, the main separation process involves electrostatic interaction. If the analyst wishes to achieve a separation with nonpolar interactions dominating the process, then the salt concentration should be high enough to suppress electrostatic interactions and the content of organic solvent should be low enough to enable the hydrophobic properties of the stationary phase to be manifested. This whole concept has to be reconsidered if acidic polypeptides are used with silica-based sorbents or strong mixed-mode phases containing cation exchanger ligands, because electrostatic repulsion between negatively charged polypeptides and the similarly charged sorbent will occur, and thus the separation will be driven by electrophoretic and hydrophobic interactions. In this case, the salt concentration has little effect on the solute–surface interaction but nevertheless affects the magnitude of the EOF, which will decrease with increasing ionic strength. Determining the magnitude of the EOF’s contribution to the overall separation mechanism appears to be extremely complex, but the options

Sample cleanup still represents a major bottleneck to using CEC as a The net charge of a polypeptide at a particular pH is determined by the extent of ionization of the side chains on basic (e.g., Lys, Arg, and His) and acidic (e.g., Asp and Glu) amino acid residues in the primary structure and from contributions from the N-terminal (amino) and C-terminal (carboxylic) functional groups. The electrostatic (silanophilic) interactions with basic polypeptides on n-alkyl bonded silicas can easily be suppressed by increasing the ionic strength of the buffer, whereby basic polypeptides elute ahead of the EOF marker because of their dominating electrophoretic mobilities. In theory, this behavior also can be achieved when mixed-mode phases containing a strong cation exchanger are used. However, in this case, joule-heating effects may become the limiting factor because high salt concentrations are needed to suppress the electrostatic interactions. The pH, ionic strength, and the percentage of organic solvent in the eluent control the interplay of mutually synergistic interactions between analytes, the stationary phase, and the eluent, respectively, and thus govern the mode of separation (29, 31, 32). Hydrophobic interactions between the solute and the sorbent can be suppressed with buffers with a high content of organic solvent. By adjusting the composition of the eluent—and thereby fine-tuning the complex balance of kinetic and thermodynamic processes—the dominant separation processes can be varied within narrow limits, recognizing that the total suppression of one mode over another cannot usually be achieved (34). If high salt concentration and a high content of organic solvent are used, basic polypeptides are separated on n-alkyl bonded silicas in p-CEC mainly due to differences in their elec-

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available with p- and ot-CEC point out their great potential as separation technologies for the biosciences. These considerations have their origin in the mutual interaction concept of CEC, whereby each of the forces involved in the generation of the selectivity—which arises from the effects of surface interaction and electromigration—are treated from rigorous thermodynamic and physical perspectives (42).

Perspectives Although some of the features of CEC have been known for more than 25 years, this powerful separation technology is still in its infancy. We now understand the origin of the EOF in a packed capillary and its impact on the mass transfer kinetics of analytes (8, 9, 28, 34, 38–40, 42). This knowledge is the basis for designing microparticulate or continuous beds with optimum pore structure and physicochemical parameters such as pore size distribution, pore size, and pore connectivity. The next step is to understand the CEC retention mechanisms for charged analytes. Liapis and Grimes have made several predictions based on modeling studies. First, the EOF is independent of the diameter of the capillary and the interstitial channels between the particles of the packed bed until the electrical double layer collapses and the flow profile becomes parabolic. Furthermore, EOF is almost independent of packing material diameter in the 0.2- to 3.0-µm range, which results in favorable intraparticle mass transfer. In addition, the velocity profile for the fluid across the capillary is flat, resulting in low zone band broadening for analytes. The axial dispersion A term for a neutral solute is much smaller

than that obtained with a parabolic velocity profile in cap-HPLC. Finally, for particles of a certain pore size, an intraparticle EOF exists that significantly enhances intraparticle mass transfer, and this EOF increases with the average packing material pore diameter and the pore interconnectivity (8, 9, 36). These modeling predictions have to be verified by experimental data. Another challenge is to investigate the retention of selected analytes in CE, cap-HPLC, and p- or ot-CEC under identical conditions to determine the orthogonality of these techniques (30). By using a systematic approach in which experimental results from separations of basic solutes in CEC are corroborated with theoretical models, it may be possible to assess the respective contributions from electrophoretic and partitioning separation mechanisms (32). Other studies have documented the behavior of sets of charged peptides with related amino acid sequences, determining the influence of effective charge, frictional ratio, intrinsic hydrophobicity, ␨-potential, and associated electrophoretic effects on the overall retention (42, 43). Collectively, these studies have shown that the structural properties of polypeptides are displayed as patterns of retention and selectivity due to the interplay of electrophoretic migration and chromatographic interaction (32). However, application experience must be gained with real samples derived from combinatorial peptide syntheses or as part of proteomic and zeomic investigations involving the separation of complex mixtures of enzymatically derived peptides. To this end, sample cleanup still represents a major bottleneck to using CEC as a workhorse in bioanalysis. Quantitative analysis for multiple, high-throughput injection experiments demanding high reproducibility and precision is another area in which considerable challenges must be faced in the immediate future and innovative solutions need to be developed. We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft and the Australian Research Council, the technical support of Agilent Technologies (Germany), and material support from Merck KGaA (Germany) and Ferring AB (Sweden).

Klaus K. Unger, formerly a professor at Johannes Gutenberg Universität (Germany), has recently established new research laboratories in collaboration with E. Merck (Germany). His research interests include microporous and mesoporous materials, synthesis and characterization of adsorbents and catalysts, and analytical and process HPLC. Milton T. W. Hearn, director of the Centre for Bioprocess Technology and the deputy director of the Australian Centre for Research on Separation Science at Monash University, was recently appointed director-designate of the Australian Research Council Special Research Centre in Green Chemistry (also at Monash University). Hearn’s research interests include the analysis, isolation, and characterization of bioactive molecules, including peptides, proteins, and other biological compounds; proteomics; and the biophysics of protein–ligand interactions. Marion Huber, Tom P. Hennessy, and Karin Walhagen are completing or have recently completed their doctoral studies. Karin Walhagen is a staff scientist at Ferring AB (Sweden) where she works on the analysis of biopharmaceuticals. Address correspondence about this article to Hearn at [email protected].

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A Critical Appraisal of Capillary Electrochromatography - Analytical

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