Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl-acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.     

Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native beta- and gamma-cyclodextrin bound to silica (ChiraDex-beta and ChiraDex-gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl-amino acids) were resolved into their enantiomers by nonaqueous CEC (NA-CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2-(N-morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.     

Ability of porous graphitic carbon to support electroosmotic flow in capillary electrochromatography

The existence of a cathodic EOF (electroosmotic flow) in the case of a porous graphitic carbon (PGC) partially packed column has been demonstrated. Then, the ability of PGC to afford electroosmosis has been brought to the fore with a fully PGC packed column. Experimental data have shown that PGC particles are negatively charged and their electrophoretic mobility has been evaluated. In order to investigate the conditions of existence of EOF different mobile phases have been tested. An EOF occurs when the conductivity of the PGC packed column is larger than the conductivity of an empty fused-silica capillary operating in the same conditions i.e. when the PGC participates in the electric conduction. Since the local electric fields in the two segments of the column are different, an evaluation of the electroosmotic mobility is not possible and the effect of the operational parameters such as the composition of the mobile phase (acetonitrile ratio and total ionic strength) has been studied in term of electroosmotic velocity V(eo).     

Controlling retention, selectivity and magnitude of EOF by segmented monolithic columns consisting of octadecyl and naphthyl monolithic segments--applications to RP-CEC of both neutral and charged solutes

Monolithic capillaries made of two adjoining segments each filled with a different monolith were introduced for the control and manipulation of the electroosmotic flow (EOF), retention and selectivity in reversed phase-capillary electrochromatography (RP-CEC). These columns were called segmented monolithic columns (SMCs) where one segment was filled with a naphthyl methacrylate monolith (NMM) to provide hydrophobic and π-interactions, while the other segment was filled with an octadecyl acrylate monolith (ODM) to provide solely hydrophobic interaction. The ODM segment not only provided hydrophobic interactions but also functioned as the EOF accelerator segment. The average EOF of the SMC increased linearly with increasing the fractional length of the ODM segment. The neutral SMC provided a convenient way for tuning EOF, selectivity and retention in the absence of annoying electrostatic interactions and irreversible solute adsorption. The SMCs allowed the separation of a wide range of neutral solutes including polycyclic aromatic hydrocarbons (PAHs) that are difficult to separate using conventional alkyl-bonded stationary phases. In all cases, the k' of a given solute was a linear function of the fractional length of the ODM or NMM segment in the SMCs, thus facilitating the tailoring of a given SMC to solve a given separation problem. At some ODM fractional length, the fabricated SMC allowed the separation of charged solutes such as peptides and proteins that could not otherwise be achieved on a monolithic column made from NMM as an isotropic stationary phase due to the lower EOF exhibited by this monolith.     

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.     

Neutral octadecyl monolith for reversed phase capillary electrochromatography of a wide range of solutes

A neutral octadecyl monolithic (ODM) column for RP capillary electrochromatography (RP-CEC) has been developed. The ODM column was prepared by the in situ polymerization of octadecyl acrylate (ODA) as the monomer and trimethylolpropanetrimethacrylate (TRIM) as the crosslinker, in a ternary porogenic solvent containing cyclohexanol, ethylene glycol, and water. The ODM column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. It is believed that the EOF is due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting to the neutral ODM column the zeta potential necessary to support the EOF required for mass transport across the monolithic column. Furthermore, the adsorption of mobile phase ions to the neutral monolith modulated solute retention and affected the separation selectivity. The wide applications of the neutral ODM column were demonstrated by its ability to separate a wide range of small and large solutes, both neutral and charged. While the separation of the neutral solutes was based on RP retention mechanism, the charged solutes were separated on the basis of their electrophoretic mobility and hydrophobic interaction with the C18 ligands of the stationary phase. As a typical result, the neutral monolithic column was able to separate peptides quite rapidly with a separation efficiency of nearly 200,000 plates/m, and this efficiency was exploited in tryptic peptide mapping of standard proteins, e. g., lysozyme and cytochrome C, by isocratic elution.     

Preparation of capillary columns coated with linear polymer containing hydrophobic and charged groups for capillary electrochromatography

A linear polymer-coated capillary was prepared by in-capillary copolymerization of N-tert-butylacrylamide (TBAAm) with a charged monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), after the capillary pretreatment with a bifunctional reagent. The coated capillaries were applied in capillary electrochromatographic (CEC) separation of small neutral compounds. Hydrophobic groups in the linear polymer, which were immobilized onto the capillary surface, functioned as the stationary phase in reversed-phase CEC separation, and charged groups in the linear polymer generated electroosmotic flow (EOF) along the column. The coated capillaries were prepared by a simple procedure. Moreover, the reproducibility with respect to EOF rate and migration times of the solutes was excellent. The results for CEC separation of small molecules using the linear polymer-coated capillaries are presented.     

Modeling the velocity field of the electroosmotic flow in charged capillaries and in capillary columns packed with charged particles: interstitial and intraparticle velocities in capillary electrochromatography systems

Mass transfer systems based on electrokinetic phenomena (i.e., capillary electrochromatography (CEC)) have shown practical potential in becoming powerful separation methods for the biotechnology and pharmaceutical industries. A mathematical model has been constructed and solved to describe quantitatively the profiles of the electrostatic potential, pressure, and velocity of the electroosmotic flow (EOF) in charged cylindrical capillaries and in capillary columns packed with charged particles. The results obtained from model simulations (i) provide significant physical insight and understanding with regard to the velocity profile of the EOF in capillary columns packed with charged porous particles which represent systems employed in CEC, (ii) provide the physical explanation for the experimental results which indicate that the velocity of the EOF in capillary columns packed with charged porous particles is a very weak function (it is almost independent) of the diameter of the particles, and (iii) indicate that the intraparticle velocity, nu(p,i), of the EOF can be greater than zero. The intraparticle Peclet number, Pe(int rap), for lysozyme was found to be greater than unity and this intraparticle convective mass transfer mechanism could contribute significantly, if the appropriate chemistry is employed in the mobile liquid phase and in the charged porous particles, in (a) decreasing the intraparticle mass transfer resistance, (b) decreasing the dispersive mass transfer effects, and (c) increasing the intraparticle mass transfer rates so that high column efficiency and resolution can be obtained. Furthermore, the results from model simulations indicate that for a given operationally permissible value of the applied electric potential difference per unit length, Ex, high values for the average velocity of the EOF can be obtained if (1) the zeta potential, zeta(p), at the surface of the particles packed in the column has a large negative magnitude, (2) the value of the viscosity, mu, of the mobile liquid phase is low, (3) the magnitude of the dielectric constant, epsilon, of the mobile liquid phase is reasonably large, and (4) the combination of the values of the concentration, C(infinity), of the electrolyte and of the dielectric constant, epsilon, provide a thin double layer. The theoretical results for the velocity of the EOF obtained from the solution of the model presented in this work were compared with the experimental values of the velocity of the EOF obtained from a fused-silica column packed with charged porous silica C8 particles. Systems with four different particle diameters and three different concentrations of the electrolyte were considered, and the magnitude of the electric field was varied widely. The agreement between theory and experiment was found to be good.     

Capillary electrochromatography with monolithic stationary phases. II. Preparation of cationic stearyl-acrylate monoliths and their electrochromatographic characterization

A novel cationic monolithic stationary phase based on the co-polymerization of pentaerythritol diacrylate monostearate (PEDAS) with a selected quaternary amine acrylic monomer was designed for performing capillary electrochromatography at high flow velocity. While PEDAS functioned as both the ligand provider and the cross-linker, the quaternary amine acrylic monomer was introduced to control the magnitude of the electroosmotic flow (EOF). The fabrication of the cationic stearyl-acrylate monolith (designated as cationic C17 monolith) with controlled porosity was achieved by free radical polymerization using the initiator 2,2'-azobisisobutyronitrile in the presence of a ternary porogenic solvent composed of cyclohexanol, ethylene glycol and water. Four different quaternary amine acrylic monomers were investigated in order to find the optimum monomer for achieving maximum electroosmotic flow (EOF) velocity. Both photo- and thermally-initiated polymerization proved effective in producing the cationic C17 monolith, and the best monolith was achieved when [2-(acryloyloxy)ethyl]trimethyl ammonium methyl sulfate (AETA) was used as the quaternary amine acrylic monomer. Although the zeta potential of the resulting cationic C17 monolith is positive with respect to water, the magnitude and direction of the EOF was markedly affected by the nature of the electrolyte in the mobile phase. Consequently, anodal, zero or cathodal EOF was observed depending on the nature of the electrolyte, and this was attributed to the adsorption of the ionic components of the electrolyte on to the solid stationary phase, which is characterized by its amphiphilic nature consisting of C17 chains, ester functions, hydroxyl groups and quaternary amine moieties. Optimized PEDAS-AETA monoliths yielded columns with high separation efficiency and allowed rapid separations on the time scale of seconds to be achieved with short capillaries.     

Migration of neutral solutes by double stepwise gradient elution in capillary electrochromatography

Characteristics of electroosmotic flow (EOF) and the migration of neutral solutes under double stepwise gradient elution in capillary electrochromatography were studied systematically. EOF velocity proved to be the function of operation time changing with the introduction of the second mobile phase. Accordingly, the retention of components also changed. The migration of neutral solutes was studied under the following three situations; A, components eluted when the column was filled only with the first kind of mobile phase; B, solutes eluted still in the first kind of mobile phase while at that time two kinds of mobile phase coexisted in the column and C, samples eluted in the second kind of mobile phase. Equations to describe the retention times of components under these three kinds of conditions were deduced and applied to predict the retention times of 12 aromatic compounds. Relative errors between experimental and calculated values were below 5.0%, which proved the reliability of the equations. In addition, parameters that might affect the retention time of solutes, such as the transferring time of mobile phase vials, the capacity factors of components and EOF velocities two steps were studied systematically.     

Protein separation by monolithic capillary electrochromatography

This work presents the separation of model proteins by capillary electrochromatography involving a monolithic stationary phase with C4 functionality. The monolith was fabricated in UV-transparent capillaries by employing a slight modification of a recently published photopolymerization procedure. With the number of theoretical plates per column ranging between 11000 and 33000, the separation efficiency proved to be lower than capillary zone electrophoresis where plate numbers ranged between 18000 and 66000. However, higher resolution was obtained due to the additional chromatographic separation mechanism. Inter- and intra-column reproducibility were evaluated, the latter could be significantly improved when using a rinsing procedure that contained 0.05% sodium dodecylsulfate in the mobile phase. Plate heights became nearly independent of mobile phase velocities higher than 0.5 mm/s indicating that high velocities can be applied without sacrificing efficiency. Furthermore, peak heights showed a dependence on injection times. For proteins, an increase in capacity factors was found when increasing the percentage of organic solvent in the mobile phase.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Separation of acidic and basic compounds in capillary electrochromatography with polymethacrylate-based monolithic columns

Methacrylate-based monolithic columns with electroosmotic flow (EOF) or very weak EOF are prepared by in situ copolymerization in the presence of a porogen in fused-silica capillaries pretreated with a bifunctional reagent. Satisfactory separations of acidic and basic compounds on the column with EOF at either low or high pH are achieved, respectively. With sulfonic groups as dissociation functionalities, sufficient EOF mobility still remains as high as 1.74 x 10(-4) cm2 s(-1) V(-1) at low pH. Under this condition, seven acidic compounds are readily separated within 5.7 min. Moreover, at high pH, the peak shape of basic compounds is satisfactory without addition of any masking amines into running mobile phase since the secondary interaction between the basic compounds and the monolithic stationary phase are minimized at high pH. Reversed-phase mechanism for both acidic and basic compounds is observed under investigated separation conditions. In addition, possibilities of acidic and basic compound separations on a monolithic column with extremely low EOF are discussed.     

Electrochromatographic separation on a poly(dimethylsiloxane)/glass chip by integration of a capillary containing an acrylate monolithic stationary phase

In this paper, we present an approach to perform electrochromatographic separations on poly(dimethylsiloxane) chips: a fused-silica capillary containing a stationary phase was introduced directly into the chip. This approach would offer great flexibility since capillary modification methods are well known, and thus, with this kind of chip, different microfluidic devices having various functions could be prepared. Electrophoretic separations were first achieved by integrating an empty capillary into the chip to evaluate the analytical performances of this system. They were compared to those obtained with a classical chip. Finally, an electrochromatographic separation involving a capillary containing a hexyl acrylate-based monolith was performed. These preliminary results show this approach to be promising.     

毛细管电色谱和加压毛细管电色谱的进展与应用

毛细管电色谱(CEC)以内含色谱固定相的毛细管为分离柱,以电渗流为驱动力,既可以分离带电物质也可以分离中性物质。它结合了毛细管电泳和高效液相色谱两者的优点,兼具高柱效、高分辨率、高选择性和高峰容量的特点,同时具有色谱和电泳的双重分离机理。然而,“纯粹”的电色谱在实际应用中有着天然的弱点,即: 在电流通过毛细管柱中的流动相时容易产生气泡(焦耳热作用),从而使电流中断和电渗流停止,毛细管柱必须被重新用流动相润湿后方能再次使用。加压毛细管电色谱(pCEC)将液相色谱中的压力流引入CEC系统中,不仅解决了气泡、干柱等问题,而且实现了定量阀进样和二元梯度洗脱。CEC和pCEC作为微分离领域的两种前沿技术,满足了当前复杂样品分析和分析仪器微型化的需求,近年来获得了广泛的关注。本文综述了这两种技术近来的发展,包括仪器、色谱固定相的发展,总结了其在生命科学、药物分析、食品安全以及环保样品分析等方面的应用进展,评述了各方法的特点,并展望了CEC和pCEC今后的发展和应用前景。     

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.     

Poly(carboxyethyl acrylate-co-ethylene glycol dimethacrylate) precursor monolith with bonded octadecyl ligands for use in reversed-phase capillary electrochromatography

A carboxy precursor monolithic column, namely poly(carboxy ethyl acrylate-co-ethylene glycol dimethacrylate) was first produced in a 100 μm i.d. fused-silica capillary and subsequently surface bonded with n-octadecyl (C₁₈) ligands by a post-polymerization functionalization process with octadecylamine in the presence of N,N´-dicyclohexylcarbodiimide. The bonding of octadecyl ligands was achieved via an amide linkage between the carboxy functions of the precursor monolith and the amino group of the octadecylamine compound. The resulting C₁₈ monolith exhibited a very low electroosmotic flow (EOF), a fact that required the incorporation of small amounts of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) in the polymerization solution to produce a precursor monolith with fixed negative charges of sulfonate groups. This may indicate that the conjugation of the carboxy functions with octadecylamine occurred to a large extent so that the amount of residual carboxy functions was sparsely dispersed and not enough to produce a desirable EOF. The EOF velocity of the C₁₈ column having fixed negative charges provided by the incorporated AMPS increased with increasing ACN content of the mobile phase signaling an increased binding of mobile phase ions to the polar amide linkages near the monolithic surface, and a decreased viscosity of the mobile phase, both of which would result in increased EOF velocity. The C₁₈ monolithic column constituted a novel nonpolar sorbent for reversed-phase capillary electrochromatography for nonpolar solutes, e.g., alkylbenzenes, alkylphenyl ketones, and polyaromatic hydrocarbons, and slightly polar compounds including phenol and chlorophenols. The C₁₈ monolithic column exhibited relatively high selectivity toward chlorophenols differing by one chloro substituent.     

Chip electrochromatography

Electrochromatography (EC) in microfluidic chips is emerging as an attractive alternative to capillary electrophoresis (CE) for on-chip separations. This review summarizes recent developments in the rapidly growing area of chip electrochromatography with a focus on "column" technologies. Relevant achievements are summarized according to the types of stationary phase used for the separations including open channels, microfabricated structures, and channels packed with beads or containing a porous monolith. The advantages and disadvantages of each, as well as practical aspects of their application, are discussed. The analytical performance of these devices is demonstrated with separations involving various families of compounds mostly in the reversed-phase chromatographic mode.     

Fluid dynamics in capillary and chip electrochromatography

This review is concerned with the phenomenological fluid dynamics in capillary and chip electrochromatography (EC) using high-surface-area random porous media as stationary phases. Specifically, the pore space morphology of packed beds and monoliths is analyzed with respect to the nonuniformity of local and macroscopic EOF, as well as the achievable separation efficiency. It is first pointed out that the pore-level velocity profile of EOF through packed beds and monoliths is generally nonuniform. This contrasts with the plug-like EOF profile in a single homogeneous channel and is caused by a nonuniform distribution of the local electrical field strength in porous media due to the continuously converging and diverging pores. Wall effects of geometrical and electrokinetic nature form another origin for EOF nonuniformities in packed beds which are caused by packing hard particles against a hard wall with different zeta potential. The influence of the resulting, systematic porosity fluctuations close to the confining wall over a distance of a few particle diameters becomes aggravated at low column-to-particle diameter ratio. Due to the hierarchical structure of the pore space in packed beds and silica-based monoliths which are characterized by discrete intraparticle (intraskeleton) mesoporous and interparticle (interskeleton) macroporous spatial domains, charge-selective transport prevails within the porous particles and the monolith skeleton under most general conditions. It forms the basis for electrical field-induced concentration polarization (CP). Simultaneously, a finite and -- depending on morphology -- often significant perfusive EOF is realized in these hierarchically structured materials. The data collected in this review show that the existence of CP and its relative intensity compared to perfusive EOF form fundamental ingredients which tune the fluid dynamics in EC employing monoliths and packed beds as stationary phases. This addresses the (electro)hydrodynamics, associated hydrodynamic dispersion, as well as the migration and retention of charged analytes.     

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography.

Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.