Streaming potential in open and packed fused-silica capillaries

The surface properties of novel stationary phases in packed and open tubular columns for capillary electrochromatography (CEC) were examined by measuring the streaming potential in a home made apparatus. The surfaces investigated include materials such as porous styrenic sorbents and octadecyl-silica as well as fused-silica tubing, in both raw and surface modified forms. Functionalization of the surface was carried out, for instance, by reductive amination or organosilane grafting on to capillary inner wall. The dependence of the streaming potential on pH was examined with aqueous solutions in the pH range from 2.5 to 9.0. Electrokinetic properties of 50 microm I.D. fused-silica capillaries have been determined by both streaming potential and electrosmotic flow measurements. Both methods gave similar pH profiles of the zeta-potential and the isoelectric points. This confirms the viability of our approach to evaluate the specific charged groups of the packing which is one of the important factors influencing electrosmotic flow (EOF) velocity and protein adsorption during a chromatographic run. In addition to bare silica capillaries, styrenic monolithic columns with different surface functionalities, which have been extensively used in our laboratory for CEC separation of peptides and proteins, were employed for comparison of two methods. Plots of zeta potential as a function of percent ACN show a complex behavior, indicating that zeta potential cannot be predicted simply from binary mixture solvent properties. It is demonstrated that the evaluation of the zeta potential by the streaming potential method is nondestructive, relatively fast, without untoward effects introduced by Joule heating and yet another means for the characterization of the surfaces under conditions employed in CEC.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。     

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.     

Recent progress in enantiomer separation by capillary electrochromatography

Enantiomer separation by electrochromatography (CEC) can be performed in three modes: (i) open-tubular capillary electrochromatography (o-CEC), in which the chiral selector is physically adsorbed coated, and thermally immobilized or covalently attached to the internal capillary wall; (ii) packed capillary electrochromatography (p-CEC), in which the capillary is either filled with chiral modified silica particles or with an achiral packing material, and a chiral selector is added to the mobile phase; and (iii) monolithic (rod)-capillary electrochromatography (rod-CEC) in which the chiral stationary phase (CSP) consists of a single piece of porous solid. We present an overview on methods and new trends in the field of electrochromatographic enantiomer separation such as CEC with either nonaqueous mobile phases or stationary phases with incorporated permanent charges, or with packing beds consisting of nonporous silica particles or particles with very small internal diameters.     

Very high-pressure capillary liquid chromatography assisted by voltage

Capillary liquid chromatography at moderately high pressures and capillary electrochromatography (CEC) have been combined to drive the mobile phase through capillary columns packed with small diameter particles. In a column packed with 1.5 microm nonporous particles, linear velocities near 3mm/s were observed when combining inlet pressures of 690 bar (10,000 psi) and an applied voltage of 25 kV. Optimum linear velocity for the column was achieved using a pressure-voltage combination of 350 bar (5000 psi) and 5 kV. Separation efficiencies at near optimum linear velocity agreed with those predicted by the van Deemter equation for liquid chromatography. Retention factors were observed to decrease under pressure-voltage combination as the voltage was increased; such a behavior has been attributed to Joule heating effects.     

Influence of the unpacked section on the chromatographic performance of duplex strong anion-exchange columns in capillary electrochromatography

This work describes initial investigations of strong anion-exchange (SAX) packing materials for capillary electrochromatography (CEC). The use of SAX phases in CEC is theoretically appealing for the analysis of negatively charged species. The reversed direction of the electroosmotic flow (EOF) generated by SAX phases (in comparison to reversed phases and strong cation-exchange phases) means that negative species can migrate with the EOF, not against it, hence the analysis times, of such species should be decreased and efficiencies improved. Duplex CEC columns (the standard for instruments using UV detection) consist of a packed and an unpacked section. Using common reversed-phase packing materials the direction of the EOF in both sections is co-linear, however when normal fused-silica capillaries are packed with SAX material the direction of the EOF in the two sections oppose one another. It has been shown, using conventional duplex CEC columns and fully packed CEC-MS columns that the opposing direction of EOF causes a massive degradation in column performance. Consequentially, it is demonstrated that if the EOF in the open section of the duplex SAX column can be controlled via pH or capillary derivatisation then good, reproducible CEC can be performed on anionic species using SAX packed CEC columns.     

Novel general expressions that describe the behavior of the height equivalent of a theoretical plate in chromatographic systems involving electrically-driven and pressure-driven flows

Novel general expressions are constructed and presented that describe the behavior of the height equivalent of a theoretical plate (plate height), H, as a function of the linear velocity, Vx, along the axis, x, of the column and the kinetic parameters that characterize the mass transfer and adsorption mechanisms in chromatographic columns. Open tube capillaries as well as columns packed with either non-porous or porous particles are studied. The porous particles could have unimodal or bimodal pore-size distributions and intraparticle convective fluid flow and pore diffusion are considered. The expressions for the plate height, H, presented in this work could be applicable to high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) systems, and could be used together with experimental plate height, H, versus linear velocity, Vx, data to determine the values of the parameters that characterize intraparticle convective fluid flow and pore diffusion. Furthermore, chromatographic systems under unretained as well as under retained conditions are examined. The experimental values of the plate height, H, versus the linear velocity, Vx, for a CEC system involving charged porous silica C8 particles and an uncharged analyte are compared with the theoretical results for the plate height, H, obtained from the expressions presented in this work. The agreement between theory and experiment is good, and the results indicate that the magnitude of the intraparticle electroosmotic flow (EOF) in the pores of the particles is substantial while the pore diffusion coefficient was of small magnitude. But the overall intraparticle mass transfer resistance in these particles was low because of the significant contribution of the intraparticle EOF. Simulation results are also presented (i) for a hybrid HPLC-CEC system, and (ii) for different CEC systems involving open capillaries as well as packed columns having non-porous or porous particles. The analysis of the results indicates (a) the reasons for the superior performance exhibited by the hybrid HPLC-CEC system over the performance obtained when the system is operated only in the HPLC mode, and (b) the operational configuration and the properties that the structure of the porous particles would have to have in CEC systems involving uncharged or charged analytes under unretained or retained conditions in order to obtain high CEC efficiency (low values of the plate height, H).     

Packing capilllary electrochromatography columns using vacuum--a preliminary study

This paper introducesa novel method for packing Capillary Electrochromatography Columns (CEC). Using vacuum packing methodology, silica particles as small as 1 microm were successfully packed into the capillary columns with 75 microm inner diameter. The columns are verystable and show no noticeable loss in efficiency after 200 sample injections. The performance of these vacuum packed capillary columns was evaluated with a mixture of aromatic and non-aromatic compounds. A 24 cm long capillary column can produce peak efficiencies of around 45,000 plates for benzene.     

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.     

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 segmented capillaries for controlling electroosmotic flow

Capillaries consisting of two segments each packed with a different stationary phase were introduced for the control and manipulation of the electroosmotic flow (EOF) in capillary electrochromatography (CEC). This kind of column configuration was called segmented capillary where one segment was packed with octadecyl silica (ODS) and served as the separation segment while the other segment was packed with bare silica and functioned as the EOF accelerator segment. The average flow in the segmented capillary increased linearly with increasing fractional length of the EOF accelerator segment, and consequently the analysis time was reduced. Under a given set of conditions, the average flow can be varied over a certain range that extends from the EOF in the individual ODS capillary at the lower end to the EOF in the individual bare silica capillary at the higher end. The pore size of the bare silica in the EOF accelerator segment influenced the average flow in the segmented capillary. Because of the difference in the EOF of the individual segments, the average flow across the segmented capillary is partially degenerated from EOF to viscous flow. Furthermore, the retaining frits in CEC columns are restrictive points which slow down the average flow, thus furthering the degeneration of the flow from EOF to viscous flow. In other words, in CEC columns containing retaining frits, the flow of the mobile phase is not only based on electroosmosis but is contaminated by a viscous component.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.     

Capillary electrochromatography using continuous-bed columns of sol-gel bonded silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups

Continuous-bed columns containing sol-gel bonded 3 microm silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups (ODS/SCX) were prepared by first packing the ODS/SCX particles into a fused-silica capillary, then filling the packed capillary with a siliceous sol-gel, curing the sol-gel, and finally drying the column with supercritical carbon dioxide. The performance of the sol-gel bonded ODS/SCX columns was evaluated for capillary electrochromatography using acetonitrile aqueous mobile phase containing phosphate buffer. The columns were mechanically strong and permeable. Both EOF velocity and current increased linearly with elevation of the applied electric field strength. The EOF velocity was high at low pH and nearly constant over a range of pH 2-9. Higher buffer concentration resulted in higher current and lower EOF velocity. The acetonitrile content had no significant effect on the EOF. Without thermosetting the column, no bubble formation was noticed with currents up to 2.5 microA. The minimum plate height of a 25/34 cm x 75 microm I.D. sol-gel bonded 3 microm ODS/SCX column was 5.7 microm (1.75 x 10(5) plates per meter) at an optimum EOF velocity of 0.92 mm s(-1). Mixtures of test aromatic compounds and aromatic hydrocarbon homologues gave symmetrical peaks when using a low pH mobile phase. The retention and elution order of aromatic compounds represented a typical reversed-phase separation mechanism similar to conventional ODS columns. The run-to-run and column-to-column retention factor reproducibility was better than 2.5% and 8.0% RSD, respectively.     

Electrochromatography with a 2.7 mm inner diameter monolithic column

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. 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.     

Preparation of fritless capillary using avidin immobilized magnetic particles for electrochromatographic chiral separation

In capillary electrochromatography (CEC), magnetic particles (MPs) were packed in a fused silica capillary by using the magnetic field to be retained without frits. For a chiral CEC separation, avidin was immobilized onto the surface of the MPs (AVI-MPs) as a stationary phase by using the physical adsorption technique. The injected AVI-MPs into the capillary were stably captured with the magnet (surface magnetic flux density, 250 mT) under the separation voltage of 10 kV (190 V/cm). By employing the fritless AVI-MPs packed capillary, the chiral separation of ketoprofen was successfully attained with the packing length of only 5 cm. Effects of the modification condition of avidin, pH of background solution, and the packing length on the enantioseparation were also investigated. Under the optimal condition, furthermore, the repeatability for the retention time of ketoprofen was better than 1.5% in the relative standard deviation and the capillary-to-capillary reproducibility was also acceptable in the prepared fritless capillaries.