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.     

Characterisation of electroosmotic flow in capillary electrochromatography columns

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.     

Enhancement of electroosmotic flow in capillary electrochromatography.

A major impediment to enhancing the speed of separation in capillary electrochromatography (CEC) is the upper limit on the electroosmotic flow (EOF) velocity by the maximal zeta potential of the chromatographic surface. Here, a new approach to speeding up EOF, suggested by Yang and El Rassi (Electrophoresis 1999, 20,18-23), is examined critically. It entails the use of a tandem arrangement of a separating column and an auxiliary column, the sole function of which is to boost EOF velocity in the separating column and thus facilitate faster analysis by CEC. Based on the principle of conservation of mass and current and using experimental data obtained in a wide range of conditions, the flow velocities in the separating and auxiliary columns were evaluated. The results show that an equidiameter open tubular auxiliary column offers a greater enhancement of EOF velocity than a packed column. Nevertheless, within the scope of the experiments the enhancement of EOF velocity by as much as 50% by using open tubular auxiliary columns has been obtained.     

Stationary phase structures enhancing electroosmotic flow in capillary electrochromatography

Several chemically bonded silicas with C18 groups were examined with respect to electroosmotic flow (EOF) velocities under CEC conditions. Stationary phases with low hydrophobic selectivity generally provided high EOFs. The stationary phases prepared by using octadecyltrichlorosilane showed greater EOF than those from octadecyldimethylchlorosilane. Restricted-access reversed-phase (RARP) packing materials having C18 groups inside the pores and silanols on the external surfaces showed higher EOF than monomeric C18 phases with similarly high hydrophobic selectivity. The RARP-type structure having silanols at the external surface seems to be effective for increasing EOF while maintaining the hydrophobic character of the solute binding sites.     

Analysis of flavonoids in leaves of Adinandra nitida by capillary electrochromatography on monolithic columns with stepwise gradient elution

Extracts of Adinandra nitida leaves, known as Shiyacha in China, were analyzed by monolithic columns of capillary electrochromatography (CEC). To obtain good resolution within a short time, stepwise gradient elution of CEC was employed, and the effects of experimental parameters, such as the buffer, the gradient conditions, and the mode of injection were studied systematically. Under optimized conditions, analysis could be accomplished in 25 min on a monolithic rod of macroporous poly(butyl methacrylate-co-ethylene dimethacrylate). With two identified flavonoids, epicatechin and apigenin, as markers, a quality control method for Shiyacha and its relevant products was established. The calibration curves exhibited good linear behavior over the concentration range of two orders of magnitude. On combination with an on-line concentration technique, the detection limit of flavonoids could be decreased to 25 ng for apigenin.     

Theory of electroosmotic flow,retention and separation efficiency in capillary electrochromatography

This article reviews recent progress that has taken place in the past decade (1992-2002) in the areas of understanding of flow of ions, buffers, as well as neutral and charged sample components through open channels and porous media. Though, primarily dealing with open capillaries and packed columns that are often used in capillary electrochromatography (CEC), these fundamental concepts are applicable to the evolving area of separations on a microchip.     

动态磁涂覆混合固定相开管毛细管电色谱中电渗流的特征

将表面分别被改性成C18和氨基的磁性纳米颗粒按照不同比例混合,制备成具有不同分离选择性的混合固定相,进一步采用动态磁涂覆的方法制备开管毛细管电色谱柱。通过考察这种色谱柱中不同种类固定相表面物理化学性质对电渗流的综合影响,从理论上说明了其电渗流的特征。分别在不同固定相配比及不同涂覆长度条件下进行实验,理论与实验结果相符,证实通过调节固定相配比或磁铁对数可以便捷地调节电渗流的大小。     

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).     

Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol-gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.     

Preparation of a neutral porous monolith and its evaluation in pressurized capillary electrochromatography with neutral and charged solutes

A neutral, nonpolar monolithic capillary column was evaluated as a hydrophobic stationary phase in pressurized CEC system for neutral, acidic and basic solutes. The monolith was prepared by in situ copolymerization of octadecyl methacrylate and ethylene dimethacrylate in a binary porogenic solvent consisting of cyclohexanol/1,4‐butanediol. EOF in this hydrophobic monolithic column was poor; even the pH value of the mobile phase was high. Because of the absence of fixed charges, the monolithic capillary column was free of electrostatic interactions with charged solutes. Separations of neutral solutes were based on the hydrophobic mechanism with the pressure as the driving force. The acidic and basic solutes were separated under pressurized CEC mode with the pressure and electrophoretic mobility as the driving force. The separation selectivity of charged solutes were based on their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase, and no obvious peak tailing for basic analytes was observed. Effects of the mobile phase compositions on the retention of acidic compounds were also investigated. Under optimized conditions, high plate counts reaching 82 000 plates/m for neutral compounds, 134 000 plates/m for acid compounds and 150 000 plates/m for basic compounds were readily obtained.     

Retention behavior of neutral solutes in pressurized flow-driven capillary electrochromatography using an ODS column

Several alkyl benzenes are separated by pressurized flow-driven capillary electrochromatography using a temperature-controlled capillary column packed with octadecyl siloxane-modified silica gel, and the effect of applied voltage on the retention is investigated. The van't Hoff plot shows good linearity at the column temperature between 305 and 330 K under applications from -6 to +6 kV. The applied voltage causes a relatively large variation in the enthalpy and the entropy of transfer of the solute from the mobile phase to the stationary phase (> 20%). However, the direction of variation in the enthalpy is almost opposite to that in the entropy, both of which might compensate each other. Therefore, the retention factor is not significantly varied (< 4%) by the application of voltage.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Separation of basic solutes by reversed-phase capillary electrochromatography

The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from -1 to infinity.     

Perfusive flow and intraparticle distribution of a neutral analyte in capillary electrochromatography

The relevance and magnitude of an electroosmotic perfusion mechanism in electrochromatography is analyzed. To systemize our studies we first considered the transport of an electroneutral and nonadsorbing tracer. Based on the refractive index matching in a microfluidic setup containing fixed spherical porous particles, we conducted a quantitative analysis in real time of the spatio-temporal distribution of fluorescent tracer molecules during their uptake by (and a release from) single particles using confocal laser scanning microscopy. Even under conditions of a significant electrical double layer overlap the intraparticle electroosmotic flow produces due to its unidirectional nature and in striking contrast to the symmetric (spherical) distributions typical for purely diffusive transport strongly asymmetric concentration profiles inside spherical particles as the locally charged pore liquid begins to respond to the externally applied electrical field. The profiles retain an axisymmetric nature, i.e., rotational symmetry with respect to the field direction. Results of our measurements could be successfully interpreted and further analyzed by a compact mathematical model. Intraparticle Peclet numbers of up to 150 have been realized and found to significantly enhance the mass transport on particle scale towards the convection-dominated regime when compared to a conventional (diffusion-limited) kinetics.     

Optimization of stepwise gradient elution in columns chromatography

Summary A general equation for the final retention of a solute chromatographed under conditions of stepwise gradient elution has been derived. The elution process and the distances travelled by solutes as a function of eluent volume were simulated by computer for the optimization of stepwise gradient prorams from isocratic HPLC data. The validity of the equations was experimentally veritied.     

Optimization of stepwise gradient elution in reversed-phase chromatography

Summary Equations describing multi-step gradient elution with a mobile phase of constant composition in each step were derived. These equations useful for calculating the retention volumes in both gradient HPLC and TLC were derived on the basis of the relationship between the isocratic capacity factor and the volume fraction of the organic modifier. The validity of the equations was experimentally verified in a LiChrosorbRP-18-water/methanol system for 11 methyl- and chlorobenzenes and phenols. A satisfactory agreement between the theoretical and experimental k′ values was found.     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

Study of the electroosmotic flow as a means to propel the mobile phase in capillary electrochromatography in view of further miniaturization of capillary electrochromatography systems

In this paper, we investigate the phenomenon of electroosmosis as a means to propel a mobile phase, in particular in view of an application in microfluidic systems, which are characterized by significantly smaller volumes of the reservoirs and the separation channels compared to conventional instrumentation. In the microfluidic chip, pH changes due to water electrolysis quickly showed an effect on the electroosmotic flow (EOF), which could be counteracted by either regularly exchanging or buffering the mobile phase. Surface treatment was of no effect in regard to EOF stabilization in empty channels but may have an influence in channels filled with a charged monolith. In fused-silica capillaries the EOF was generally found to decrease from 'naked' to surface-treated to monolith-filled capillaries. The EOF tended to be higher when an organic solvent (acetonitrile) was added to the mobile phase and could be further increased by substituting the water with equal amounts of methanol. In addition, the hydrostatic pressure exerted by the EOF was investigated. In a microfluidic chip with empty (cross-)channels such an effect could be responsible for a redirection of the flow. In capillaries partially filled with a noncharged (non-EOF-generating) monolith, a linear relationship could be established between the EOF created in the empty section of the capillary (apparent mobility) and the length of the monolith (backpressure). In capillaries partially filled with a charged (EOF-producing) monolith, flow inhomogeneities must be expected as a consequence of a superimposition of hydrodynamic pressure and EOF as mobile phase driving force.     

Influence of moderate Joule heating on electroosmotic flow velocity, retention, and efficiency in capillary electrochromatography

The influence of Joule heating on electroosmotic flow velocity, the retention factor of neutral analytes, and separation efficiency in capillary electrochromatography was investigated theoretically and experimentally. A plot of electrical current against the applied electrical field strength was used to evaluate the Joule heating effect. When the mobile phase concentration of Tris buffer exceeded 5.0 mM in the studied capillary electrochromatography systems using particulate and monolithic columns (with an accompanying power level of heat dissipation higher than 0.35 W/m), the Joule heating effect became clearly noticeable. Theoretical models for describing the variation of electroosmotic flow velocity with increasing applied field strength and the change of retention factors for neutral analytes with electrical field strength at higher Tris buffer concentrations were analyzed to explain consequences of Joule heating in capillary electrochromatography. Qualitative agreement between experimental data and implications of the theoretical model analysis was observed. The decrease of separation efficiency in capillary electrochromatography with macroporous octadecylsilica particles at high buffer concentration can be also attributed to Joule heating mainly via the increased axial diffusion of the analyte molecules and dispersion of solute bands by a nonuniform electroosmotic flow profile over the column cross-section. However, within a moderate temperature range, the contribution of the macroscopic velocity profile in the column arising from radial temperature gradients is insignificant.     

Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance

Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous-flow CEC-NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE-NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two-dimensional (2-D) spectrum of the synthetic peptide aspartame.