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.     

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.     

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.     

Use of mixed-mode sorbents for the electrochromatographic separation of thrombin receptor antagonistic peptides

In this study, the thrombin receptor antagonistic peptide TRAP-1 and its alanine-scan analogues, TRAP 2-6, have been employed as probes to characterise the performance of C18/SCX mixed-mode capillary electrochromatographic (CEC) columns. It was found that the resolution of this group of peptides could only be achieved in a narrow pH range with phosphate-based running electrolytes. The influence of the running electrolyte composition, e.g. the buffer choice, the ionic strength, the pH and the organic solvent content, on the electroosmotic flow (EOF) of these mixed-mode CEC columns was investigated. In addition, the retention mechanism for this group of peptide probes in the electrochromatographic process was studied by examining the effect of varying the running electrolyte composition. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving both hydrophobic as well as ion exchange interactions. By modulating the running electrolyte composition, the hydrophobic or ion exchange components of the interaction process could be made to dominate the chromatographic retention of the peptides. Based on this strategy, a high-resolution separation of six closely related synthetic peptides was demonstrated with this mixed-mode CEC system.     

Capillary electrochromatography of peptides on a column packed with tentacular weak cation-exchanger particles

Silica-based, tentacular weak cation-exchanger particles were prepared for use as the stationary phase in the separation of positively charged sample components by capillary electrochromatography (CEC). Silica beads were first silanized with 3-(trimethoxysilyl) propyl methacrylate that served as a heterobifunctional linker, which reacted with 2-acrylarmidoglycolic acid in a second step by radical polymerization in aqueous solution. Baseline separation of basic peptides with good column efficiency was obtained on packed capillary columns by isocratic elution CEC with NaCl as the mobile phase modulator. The retention mechanism in the electrochromatographic process was studied by examining the effect of salt concentration on the migration behavior of the peptides. The chromatographic retention factor k'(lc) for charged sample components in the electrochromatographic process was estimated on the assumption that the overall migration rate of a charged migrant can be taken as the sum of the rate of chromatographic elution and the rate of electrophoretic migration. The estimated k(lc) values from experimental results were plotted against the molal salt concentration on a double logarithmic scale. The linear correlation is in good agreement with the prediction by the theory on the basis of traditional ion-exchange chromatography. The comparison of CEC results, obtained with open tubular and packed capillary columns having the same retentive functions as the stationary phase, supports the notion that variation of the phase ratio in the column offers an additional means to modulate the electrochromatographic migration behavior.     

Toward rapid preparation of capillary columns for electrochromatography use

CEC is a high performance electrodriving liquid phase separation technique. It does not need complex and sophisticated high pressure instrumentation for nanoflow driving. This is attractive for parallel multicolumn analysis. To this end, high throughput methods for column preparation are needed to support the use of multiple columns. In this study, we directly used CEC mobile phase solution as the packing solvent, and realized rapid preparation of capillary columns based on a single particle fritting technology. The method presented high preparation throughput compared with other reported methods based on various fritting technologies. The single particle fritting approach promoted column preparation throughput to 1 column/h, including all the fritting, packing and conditioning steps. The rapidly prepared columns showed consistently high efficiency of up to 150 000 plates per meter, and usefulness in reversed phase CEC of neutral, charged and biomolecules. With standard peptides as the sample, excellent long term reproducibility (better than 0.8%RSD, ten days, for retention times) was observed.     

Capillary electrochromatography of peptides in a microfabricated system.

Reversed-phase liquid chromatography of tryptic peptides is shown in the capillary electrochromatography mode using microfabricated columns. Although selectivity is different, a mixture of tryptic peptides from ovalbumin appears to be as easily separated in the CEC as HPLC mode. The major difference between a separation in the macrofabricated CEC column and conventional separations in the HPLC mode is that separations are more readily achieved in the isocratic mode in the lower surface area microfabricated CEC columns.     

Pressure‐assisted CEC versus CEC using methacrylate‐based monolithic columns: Influence of the polymerization‐mixture composition

Pressure‐assisted CEC (pCEC) can either be performed on a CE instrument by adding pressure at the column inlet, or by applying voltage on a capillary liquid chromatography system. This study investigates the pressure's added value in pCEC using an LC instrument as well as the influence of the polymerization‐mixture composition on monolithic columns in such experimental circumstances. Two factors of the polymerization mixture, which is used to prepare the monolithic capillary columns, were varied according to an experimental design approach: the pore‐forming solvent/total monomer ratio and the pore‐forming solvents composition. Initially, the effect of the resulting stationary phase on the elution behavior of mainly pharmaceutical compounds was studied. Four responses were used to evaluate the effects on the chromatography: retention time, retention factor, peak asymmetry and number of theoretical plates. After processing the results, the stationary phase composition with the best chromatographic behavior was determined and tested. The advantageous properties of this stationary phase compared with the design center‐point column were demonstrated. Secondly, the results of these pCEC experiments were compared with those generated in an identical experimental setup previously performed using CEC. Chromatographic conditions were chosen so that the center‐point column showed similar retention in CEC and pCEC. The expected advantage (faster analysis) and drawback (decreased efficiency) of pCEC in the analysis of pharmaceuticals was evaluated. Analysis time and efficiency were both found to depend greatly on the porosity of the column. The conclusion of this comparison is that pCEC did not have a significant added value to CEC. However, this was mainly due to the instrument's limitation of the pressure‐driven flow over the column. A clear benefit of the pCEC setup was apparatus‐related, i.e. the presence of a loop injection system on the pCEC instrument, which avoids the injection problems that were occasionally observed in CEC.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.     

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.     

Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as separation media for electrochromatography

Photoinitiated free radical polymerization has been used for the preparation of porous polymer monoliths within UV transparent fused silica capillaries and quartz tubes. These formats were used as models for the preparation of the separation media within channels of microfabricated devices. A mixture of ethylene dimethacrylate, butyl methacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid was polymerized in the presence of a porogenic solvent consisting of 1-propanol, 1,4-butanediol, and water at room temperature under UV irradiation. Modification of the porogen composition enables the tailoring of pore size within the broad range from ca. 100 to 4000 nm. Scanning electron micrographs confirmed the homogeneity of the porous structure of the materials prepared, even in a quartz tube with a diameter as large as 4 mm. Separation properties of the resulting capillary columns were tested in capillary electrochromatography (CEC) mode using a mixture of thiourea and eight aromatic compounds. Plate number as high as 210 000 plates/m were found for a capillary column with optimized porous properties. The monolithic columns were also able to separate mixtures of peptides.     

A novel open-tubular capillary electrochromatography with magnetic nanoparticle coating as stationary phase

A novel open‐tubular capillary electrochromatography (OT‐CEC) with modified core/shell magnetic nanoparticles coating as stationary phase was introduced using external magnetic force to fix magnetic nanoparticles. The magnetic nanoparticles coating inside the capillary columns could be easily regenerated by removing and re‐applying the external magnetic field. Magnetic field intensity, concentration and flow rate of nanoparticles suspension were investigated to achieve simple and stable preparation. Mixture of five organic acids was used as the marker sample to evaluate the OT‐CEC system, and the relative column efficiency of anthranilic acid reaches 220 000 plates/m. The excellent within‐column and between‐column repeatability has been testified with the RSDs of retention time of less than 1.51 and 5.29%, respectively. The aqueous extract of rhizoma gastrodiae was analyzed by the OT‐CEC system, and 23 peaks were eluted in 30 min. Compared with conventional open‐tubular capillary column, this new system shows faster separation speed and higher column efficiency from the larger surface area of nanoparticles. It has great potential in the method development for the analysis of complex samples, since magnetic coating can effectively prolong the column life by expediently replacing stationary phase to eliminate the pollution or irreversible adsorption.     

Recent advances in the control of morphology and surface chemistry of porous polymer-based monolithic stationary phases and their application in CEC

This review focuses on developments in the field of polymer-based monolithic columns for CEC published in the literature since the beginning of the year 2005. The possibility of in-situ preparation as well as easy control over their porous properties and surface chemistries clearly make monolithic separation media an attractive alternative to capillary columns packed with particles. Different variables such as polymerization conditions, morphology, and surface chemistry are shown to directly affect performance of monolithic capillary columns in terms of efficiency, analysis time, and retention.     

Capillary electrochromatography of proteins and peptides

This review summarizes applications of CEC for the analysis of proteins and peptides. This "hybrid" technique is useful for the analysis of a broad spectrum of proteins and peptides and is a complementary approach to liquid chromatographic and capillary electrophoretic analysis. All modes of CEC are described--granular packed columns, monolithic stationary phases as well as open-tubular CEC. Attention is also paid to pressurized CEC and the chip-based platform.     

Alkylthiol gold nanoparticles in open-tubular capillary electrochromatography

Open-tubular columns for capillary electrochromatography (CEC) were formed by immobilising dodecanethiol gold nanoparticles on prederivatised 3-aminopropyl-trimethoxysilane (APTMS) or 3-mercaptopropyl-trimethoxysilane (MPTMS) fused-silica capillaries. The initial stage of this research involved the synthesis and characterisation of dodecanethiol gold nanoparticles, with tunnelling electron microscopy analysis of the dispersed phase of the gold nanoparticles dispersion in CHCl3, revealing spherical particles. The surface features of an Au-MPTMS coated capillary column were determined using scanning electron microscopy. The electroosmotic flow characteristics of Au-APTMS and Au-MPTMS capillary columns were then determined, by varying the pH and the voltage. The electrochromatographic properties of the gold nanoparticles CEC capillaries were investigated using a "reversed-phase" test mixture of thiourea, benzophenone and biphenyl and selected pyrethroid pesticides. Efficient separations of benzophenone and biphenyl solutes on Au-MPTMS and Au-APTMS capillary columns were obtained, as were linear plots of logarithm capacity factor versus % MeOH. A study of the reproducibility of retention for these solutes on Au-APTMS, Au-MPTMS and on a loosely coated capillary demonstrated the necessity of a coupling agent to prevent the gold nanoparticles from washing-off. These dodecanethiol gold capillary columns are easier to produce and operate than packed capillary columns. The research work confirms the use of gold nanoparticles as a novel phase for open-tubular CEC, demonstrating reproducible retention and characteristic reversed-phase behaviour.     

毛细管电色谱柱重复性考察及实验条件的选择

成功地研制了７５μｍ内径偶联式和７５,１００μｍ内径非偶联式毛细管电色谱柱。在自制电色谱柱上获得了小于２．１２％的保留时间ＲＳＤ值,考察了ｐＨ值、有机溶剂浓度对保留行为的影响,并用以指导实验条件的选择。比较了两种方式电色谱柱的峰形。     

Measurement of Henry coefficients by capillary columns coated with slightly polar solvents

Summary The specific retention volumes and Henry coefficients measured on packed columns are compared with those calculated via retention indices determined on wall coated open tubular capillary columns. The stationary phase liquids used were derivatives of a branched paraffin, C₇₈H₁₅₈, where a methyl or ethyl group of the paraffin was replaced by OH, CN, OCH₃ and SH groups. The retention indices of a series of molecular probes of varying polarity (aromatics, 1-chloroalkanes, 1-acetoxy-alkanes, 1-alkanols, 2-alkanones) were determined atT=403.15 K on fused-silica open tubular columns prepared by static coating that gave stable and uniform solvent films. After obtaining the absolute data on packed columns forn-alkanes, the specific retention volumes and Henry coefficients of the solutes were calculated. The retention data obtained on both columns showed good agreement. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Some considerations concerning the composition of the mobile phase in capillary electrochromatography

In capillary electrochromatography (CEC) the propulsion of the mobile phase is effected by electroosmosis. The velocity of the electroosmotic flow is dependent on surface properties of the stationary phase and on bulk properties of the mobile phase. Therefore, in CEC the optimization of the mobile phase composition must take more factors into account than in pressure-driven LC. In this paper, the impact of the electrolyte concentration in the mobile phase and of the volume fraction of the organic mobile phase constituent on the velocity of the electroosmotic flow and on the chromatographic efficiency is investigated for CEC with capillaries packed with octadecylsilica gel. Bias of the data by an open section of the capillary has been excluded by employing completely packed capillaries and detection in a packed section. Acetonitrile as organic constituent of the mobile phase is compared to other possible organic modifiers (polar organic solvents) concerning influence on velocity of the electroosmotic flow and retention of solutes.     

Macroporous polymeric monoliths as stationary phases in gas adsorption chromatography

The influence of the conditions of preparation of divinylbenzene-based polymer monoliths on the properties of monolithic capillary columns for use in gas adsorption chromatography was examined. It was found that the polymerization time and the temperature and composition of the polymerization mixture have an effect on the dynamic and chromatographic properties of the columns. The monolithic capillary columns prepared under the optimal synthesis conditions had the height equivalent of a theoretical plate at the level of 20–30 μm, which is an order of magnitude below that of conventional open tubular columns.