Mass transport in a micro flow-through vial of a junction-at-the-tip capillary electrophoresis-mass spectrometry interface

When coupling capillary electrophoresis with postcolumn detection methods, such as mass spectrometry, the presence of postcolumn band broadening must be considered. The band broadening effects introduced by junction-at-the-tip CE-MS interfaces using a postcolumn micro flow-through vial are investigated by studying the hydrodynamic flow patterns and mass transport process inside the micro vial at the end of the CE separation capillary. Simulation results obtained by solving the Navier-Stokes and mass balance equations provide insights into the velocity field and concentration distribution of the analytes in the micro vial and demonstrate that, with a low flow rate of chemical modifier solution, the laminar flow streams confine the analyte molecules to the central part of the micro vial and thus maintain major features of the peak shapes. Peaks detected by UV and MS under similar experimental conditions were compared to verify the numerical prediction that the main features of the UV peak can be retained in the MS peak. Experiments also show that band broadening can be minimized when an appropriate chemical modifier flow rate is selected.     

Flow counterbalanced capillary electrophoresis using packed capillary columns: resolution of enantiomers and isotopomers

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.     

Electroosmotic capillary flow with nonuniform zeta potential

The present work is an analytical and experimental study of electroosmotic flow (EOF) in cylindrical capillaries with nonuniform wall surface charge (zeta-potential) distributions. In particular, this study investigates perturbations of electroosmotic flow in open capillaries that are due to induced pressure gradients resulting from axial variations in the wall zeta-potential. The experimental inquiry focuses on electroosmotic flow under a uniform applied field in capillaries with an EOF-suppressing polymer adsorbed onto various fractions of the total capillary length. This fractional EOF suppression was achieved by coupling capillaries with substantially different zeta-potentials. The resulting flow fields were imaged with a nonintrusive, caged-fluorescence imaging technique. Simple analytical models for the velocity field and rate of sample dispersion in capillaries with axial zeta-potential variations are presented. The resulting induced pressure gradients and the associated band-broadening effects are of particular importance to the performance of chemical and biochemical analysis systems such as capillary electrokinetic chromatography and capillary zone electrophoresis.     

Analysis of the performance of a flow reactor for use with microcolumn HPLC

Postcolumn derivatization reactions can be used to improve detector sensitivity or selectivity. The advantages of capillary chromatography for trace analysis could be augmented if there were postcolumn reactors suitable for microchromatographic systems. However, postcolumn derivatization is a challenge because of the small peak volumes associated with capillary columns. We have developed a postcolumn flow reactor from microchannels formed in fluorinated ethylene propylene and 50-microm fused-silica tubing for use with capillary HPLC analyses. Theoretical and experimental evidence show that the reactor, which operates in the Taylor dispersion regime, enables contact of analyte and derivatization streams purely by diffusion. Reactor lengths as short as 2 cm allow formation of copper(II)-peptide complexes that are detected electrochemically at a carbon fiber microelectrode. The reactor has been used with 100-microm-i.d. columns with insignificant effects (i.e., <3%) on peak band spreading. Theoretical calculations indicate that even smaller i.d. columns can be used with little effect on chromatographic resolution.     

Optimizing band width and resolution in micro-free flow electrophoresis

The broadening mechanisms for micro-free flow electrophoresis (micro-FFE) have been investigated using a van Deemter analysis. Separation power, the product of electric field and residence time, is presented as a parameter for predicting the position of sample streams and for comparing separations under different conditions. Band broadening in micro-FFE is governed by diffusion at lower linear velocities and a migration distance-dependent mechanism at higher linear velocities. At higher linear velocities, the parabolic flow profile is elongated, generating a distribution of analyte residence times in the separation channel. This distribution of residence times gives rise to a distribution of migration distances in the lateral direction since analytes spend different amounts of time in the electric field. Equations were derived to predict the effect of electric field and buffer flow rate on broadening. Experimental data were collected to determine whether the derived equations were useful in explaining broadening caused by diffusion and hydrodynamic flow at different linear velocities and electric fields. Overall there was an excellent correlation between the predicted and experimentally observed values allowing linear velocity and electric field to be optimized. Suppression of electroosmotic flow is proposed as a means of reducing micro-FFE band broadening due to hydrodynamic effects and maximizing resolution and peak capacity.     

Porous polymer monolith assisted electrospray

Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-microm i.d.) at a variety of flow rates (50-1000 nL/min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (<100 nL/min) a mist, presumably from multiple small Taylor cones, develops. The hydrophobic nature of the PPM should limit problems with band broadening associated with droplet spreading at the capillary exit, while the multiple flow paths inherent in the PPM minimize clogging problems associated with conventional nanospray emitters. Total ion current traces for a constant infusion of standard PPG and cytochrome c solutions are very stable with deviations ranging from only 3 to 8%. The PPM-assisted electrospray produces mass spectra with excellent signal-to-noise ratios from only a few femtomoles of material.     

Effect of analyte adsorption on the electroosmotic flow in microfluidic channels

The predictability and constancy over time of the electroosmotic flow in microchannels is an important consideration in microfluidic devices. A common cause for alteration of the flow is the adsorption of analytes to channel walls, for example, during capillary electrophoresis of proteins. It is shown that certain experimental data, published by Towns and Regnier (Towns, J. K; Regnier, F. E. Anal. Chem. 1992, 64, 2473-2478.), on the anomalous elution times for proteins in capillary electrophoresis can be explained using a simple model for analyte adsorption that uses a result first reported by Anderson and Idol (Anderson, J. L.; Idol, W. K Chem. Eng. Commun. 1985, 38, 93-106.) on the electroosmotic flux in capillaries with axial variations in zeta-potential. It is suggested that it might be possible to use such a model to dynamically correct for altered elution times in capillary electrophoretic devices.     

Fast, high peak capacity separations in gas chromatography-time-of-flight mass spectrometry

Peak capacity production (i.e., peak capacity per separation run time) is substantially improved for gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and applied to the fast separation of complex samples. The increase in peak capacity production is achieved by selecting appropriate experimental conditions based on theoretical modeling of on-column band broadening, and by reducing the injection pulse width. Modeling to estimate the on-column band broadening from experimental parameters provided insight for the potential of achieving GC separations in the absence of off-column band broadening, i.e., the additional band broadening not due to the on-column separation process. To optimize GC-TOFMS separations collected with a commercial instrumental platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a commercially available thermal modulator is adapted and applied (referred to herein as thermal injection) to provide a narrow injection pulse, while the TOFMS provided a data collection rate of 500 Hz, initially averaged to 100 Hz for data storage. The use of long, relatively narrow open tubular capillary columns and a 30 °C/min programming rate were explored for GC-TOFMS, specifically a 20 m, 100 μm inner diameter (i.d.) capillary column with a 0.4 μm film thickness to benefit column capacity, operated slightly below the optimal average linear gas velocity (at ~2 mL/min, due to the flow rate constraint of the TOFMS). Standard autoinjection with a 1:100 split resulted in an average peak width of ~1.2 s, hence a peak capacity production of 50 peaks/min. Metabolites in the headspace of urine were sampled by solid-phase microextraction (SPME), followed by thermal injection and a ~7 min GC separation (with a ~6 min separation time window), producing ~660 ms peak widths on average, resulting in a total peak capacity of ~550 peaks (at unit resolution) and a peak capacity production of ~90 peaks/min (~2-fold improvement relative to standard autoinjection with the 1:100 split). This total peak capacity production achieved is equivalent to, or greater than, that currently utilized in metabolomics studies using GC/MS, but with much slower separations, on the order of 40 to 60 min, corresponding to a 5-fold or greater GC/MS analysis throughput rate.     

Mathematical model describing gradient focusing methods for trace analytes

The problem of gradient focusing for concentrating trace analytes is considered. Variation of buffer viscosity, conductivity, and possibly also the zeta-potential results in a focusing point where the electrophoretic velocity is balanced by the electroosmotic flow (EOF) and where the sample concentrates. The axial inhomogeneity also results in an induced pressure gradient that alters the EOF profile and therefore causes Taylor dispersion. The coupled hydrodynamics and transport problem leading to the achievement of a steady state is studied in the context of the lubrication approximation: all variations in the axial direction take place over a length scale very much larger than the characteristic channel width. A single length scale sigma(m) and a single time scale tau is found to completely determine the dynamics of the evolution close to the focusing point. Using appropriate scaled variables, the time evolution of the concentration profile near equilibrium can be described by an inhomogeneous advection diffusion equation that is free of all parameters. Explicit formulas are deduced for the location of the peak centroid and its width as a function of time. A simple graphical method is proposed for optimizing the performance of the system when some tunable external parameters are available.     

Effects of pore flow on the separation efficiency in capillary electrochromatography with porous particles

The effect of pore flow on the separation efficiency of capillary electrochromatography (CEC) has been studied using columns packed with particles with different pore sizes. A previously developed model was used to predict the (relative) pore flow velocity in these columns under various experimental conditions. Equations are derived describing the effect of pore flow on peak broadening in CEC. The theory has been compared with practice in the reversed-phase CEC separation of various polyaromatic hydrocarbons. It is shown, by theory and experimentally, that the mass-transfer resistance contribution to peak dispersion can be effectively eliminated when using porous particles with a high (> or =50 nm) average pore diameter. Moreover, at high pore-to-interstitial flow ratios the flow inhomogeneity contribution (the A term in the plate height equation) is also shown to decrease. Under optimal conditions, a reduced plate height of 0.3 for the nonretained compound could be obtained. It is argued that fully perfusive porous particles can be a more efficient separation medium in CEC than nonporous particles.     

Continuous separation of high molecular weight compounds using a microliter volume free-flow electrophoresis microstructure

A microliter volume free-flow electrophoresis microstructure (μ-FFE) was used to perform a continuous separation of high molecular weight compounds. The μ-FFE microstructure had a separation bed volume of 25 μL and was fabricated from silicon using standard micromachining technology. Laser-induced fluorescence was used to detect the sample components, which were labeled with fluorescein isothiocyanate (FITC) prior to analysis. The continuous separation of human serum albumin (HSA), bradykinin, and ribonuclease A demonstrated that only 25 V/cm was required to isolate HSA from bradykinin and ribonuclease A, while 100 V/cm was needed for the separation of bradykinin from ribonuclease A. Comparison of the observed band broadening with the theoretical variance indicated that dispersion due to the initial bandwidth, diffusion, and hydrodynamic broadening were the main contributors to the band broadening of HSA and bradykinin. However, the band broadening for ribonuclease A could not be sufficiently accounted for using the above contributors. Adsorption was found to be a possible contributor to the overall variance for ribonuclease A. Calculation of the theoretical variance due to Joule heating indicated that broadening due to Joule heating effects was insignificant. This was likely due to the narrow cross-sectional area of the device, which facilitated efficient cooling. Electrohydrodynamic distortion was observed for HSA as it migrated toward the side bed. Studies of the resolution of bradykinin and ribonuclease A as a function of field strength at various sample and carrier flow rates indicated that, for maximum throughput, high field strengths and high flow rates were required. However, no optimal conditions were found. The μ-FFE device has a peak capacity of ～8 bands/cm, while for a typical separation of proteins using a commercial system, a peak capacity of 10 bands/cm is obtained. Thus, the resolving power of the μ-FFE device is similar to those of conventional systems. The continuous separation of tryptic digests of mellitin and cytochrome c demonstrated the ability to continuously separate more complex mixtures. Finally, modifications were made to the microstructure to facilitate fraction collection, and the fractionation of whole rat plasma was performed. Off-line analysis of the resulting fractions indicated that the complete isolation of serum albumin and globulins was possible using a field strength of 25 V/cm.     

Electroosmotic flow in composite microchannels and implications in microcapillary electrophoresis systems

The electroosmotic flow in laminated excimer laser-ablated microchannels has been studied as a function of the depth of the rectangular channels, and particular emphasis has been given to the difference in the zeta-potentials between the lamination layer and the ablated substrate. Experimental electroosmotic flow follows the tendency predicted by a recently published model. The zeta-potentials of lamination and ablated surfaces were determined for poly(ethylene terephthalate) and poly(carbonate) substrates by fitting the experimental data with a numerical implementation of this model. In the experimentally investigated range of channel cross sections, a linear fit to the data gives a good approximation of the zeta-potentials for both materials. Moreover, a flow injection analysis of fluorescein dye has been performed to show the severe loss in numbers of theoretical plates, caused by Taylor dispersion, when such microchannels, dedicated to microcapillary electrophoresis, are used.     

Moment analysis of mass-transfer kinetics in C18-silica monolithic columns

The moment analysis of elution peak profiles based on new moment equations provides information on the mass-transfer characteristics of C(18)-silica monolithic columns. The flow rate dependence of the HETP data was analyzed using the generalized van Deemter equation, after correction of these data by subtraction of the external mass-transfer contribution to band broadening. Kinetic parameters and diffusion coefficients related to the mass-transfer processes in monolithic columns were derived by taking advantage of the different flow velocity dependence of their contributions to band broadening. At high flow rates, axial dispersion and diffusive migration across the monolithic C(18)-silica skeleton contribute much to band broadening, suggesting that it remains important to reduce the influence of eddy diffusion and the mass-transfer resistance in the stationary phase to achieve fast separations and a high efficiency. Surface diffusion plays a predominant role for molecular migration in the monolithic stationary phase. Although the value of the surface diffusion coefficient (D(s)) depends on an estimate of the external mass-transfer coefficient, D(s) values of the order of 10(-7) cm(2) s(-1) were calculated for the first time for the C(18)-silica monolithic skeleton. The value of D(s) decreases with increasing retention of sample compounds. Analysis of a kind of time constant calculated from D(s) suggests that the "chromatographic corresponding particle size" is approximately 4 microm for the C(18)-silica monolithic stationary phase used in this study. The accuracy of the D(s) values determined was discussed.     

Diffusivity of solutes measured in glass capillaries using Taylor's analysis of dispersion and a commercial CE instrument

We present a strategy for the rapid, efficient, and accurate measurement of the coefficient of diffusion (D) of solutes using a commercial capillary electrophoresis (CE) instrument. This approach utilizes the classic analysis of Taylor of the dispersion of solutes pumped hydrostatically through glass capillaries. To obtain accurate values of D, we modified Taylor's analysis of dispersion to account for the finite time required to reach steady-state flow in the capillary when using a CE instrument. Neglecting this effect results in measured diffusivities of phenylalanine, a model solute, that are in error by as much as 60% when compared with published values. We provide an analysis of this effect and a simple strategy for avoiding these errors. Using this approach, we analyze profiles of concentration fronts and measured values of D for phenylalanine to within 5% of published values. We also analyze profiles of pulses of solute. To determine values of D accurately, measurements of dispersion first need to be made as a function of injection volume to correct for the finite width of the injection plug, before they are corrected for unsteady-state flow. This approach also yields values of D for phenylalanine to within 5% of published values. In contrast to other techniques used for the determination of D, this approach requires no fluorescent labeling and is applicable to solutes of any molecular weight.     

Imaging of electroosmotic flow in plastic microchannels

We have characterized electroosmotic flow in plastic microchannels using video imaging of caged fluorescent dye after it has been uncaged with a laser pulse. We studied flow in microchannels composed of a single material, poly(methyl methacrylate) (acrylic) or poly(dimethylsiloxane) (PDMS), as well as in hybrid microchannels composed of both materials. Plastic microchannels used in this study were fabricated by imprinting or molding using a micromachined silicon template as the stamping tool. We examined the dispersion of the uncaged dye in the plastic microchannels and compared it with results obtained in a fused-silica capillary. For PDMS microchannels, it was possible to achieve dispersion similar to that found in fused silica. For the acrylic and hybrid microchannels, we found increased dispersion due to the nonuniformity of surface charge density at the walls of the channels. In all cases, however, electroosmotic flow resulted in significantly less sample dispersion than pressure-driven flow at a similar velocity.     

Reexamination of Dependence of Plate Number on SDS Concentration in Micellar Electrokinetic Chromatography

Chromatograms of hydrophilic, hydrophobic, and intermediate-polarity analytes were developed in 50-μm capillaries by micellar electrokinetic chromatography at field strengths less than 31 kV/m. The analytes were solubilized by phosphate/borate buffers containing 15, 50, and 100 mM sodium dodecyl sulfate (SDS). The plate numbers N of the analytes, as well as those of the electroosmotic flow and micellar markers, were compared to predictions of N estimated by a simple model based on longitudinal diffusion and plug size. Good to fair agreement between theory and experiment was obtained for the hydrophilic and intermediate-polarity analytes in all buffers over the entire field strength range. Good agreement between theory and experiment was obtained for the hydrophobic analyte and micellar marker in all buffers at low field strengths; however, these compounds were subject to dispersion at higher field strengths by what appears to be Joule heating. The magnitudes of other, closely related Joule heating losses are quantified here using temperature profile measurements by Morris and co-workers and Taylor dispersion calculations. In contrast to the commonly reported increase of N with media concentration, the Ns of the hydrophilic and intermediate-polarity analytes were found to be essentially independent of SDS concentration over the investigated SDS range, and the Ns of the hydrophobic species were found to be independent of SDS concentration until (what appears to be) Joule heating became significant. These results were compared to those of Sepaniak and Cole. A critique of some previous studies of N vs SDS concentration is presented, in which quantitative explanations for some dispersions are offered as alternatives to surfactant concentration effects.     

Electroosmotic and pressure-driven flow in open and packed capillaries: velocity distributions and fluid dispersion

The flow field dynamics in open and packed segments of capillary columns has been studied by a direct motion encoding of the fluid molecules using pulsed magnetic field gradient nuclear magnetic resonance. This noninvasive method operates within a time window that allows a quantitative discrimination of electroosmotic against pressure-driven flow behavior. The inherent axial fluid flow field dispersion and characteristic length scales of either transport mode are addressed, and the results demonstrate a significant performance advantage of an electrokinetically driven mobile phase in both open-tubular and packed-bed geometries. In contrast to the parabolic velocity profile and its impact on axial dispersion characterizing laminar flow through an open cylindrical capillary, a pluglike velocity distribution of the electroosmotic flow field is revealed in capillary electrophoresis. Here, the variance of the radially averaged, axial displacement probability distributions is quantitatively explained by longitudinal molecular diffusion at the actual buffer temperature, while for Poiseuille flow, the preasymptotic regime to Taylor-Aris dispersion can be shown. Compared to creeping laminar flow through a packed bed, the increased efficiency observed in capillary electrochromatography is related to the superior characteristics of the electroosmotic flow profile over any length scale in the interstitial pore space and to the origin, spatial dimension, and hydrodynamics of the stagnant fluid on the support particles' external surface. Using the Knox equation to analyze the axial plate height data, an eddy dispersion term smaller by a factor of almost 2.5 than in capillary high-performance liquid chromatography is revealed for the electroosmotic flow field in the same column.     

Method for the elimination of chromatographic bias from measured capillary electrophoretic effective mobility values

A method based on a modified version of pressure-mediated capillary electrophoresis (PreMCE) has been developed for the elimination of the chromatographic bias inherent in effective electrophoretic mobilities measured by capillary electrophoresis. This new five-band PreMCE method can be readily executed on most commercial capillary electrophoresis instruments. It yields not only precise but also accurate effective mobilities and electroosmotic flow rates, even when the analytes and electroosmotic flow markers are strongly retained on the coated fused-silica capillary wall.     

Turn geometry for minimizing band broadening in microfabricated capillary electrophoresis channels

Turns in microfabricated capillary electrophoresis channels generally result in degraded separation quality. To circumvent this limitation, channels were constructed with different types of turns to determine the design that minimizes turn-induced band broadening. In particular, tapered turns were created by narrowing the separation channel width before the start of a turn and widening the channel after the turn is complete. The radius of curvature of the turn, the length over which the channel is tapered, and the degree of tapering were explored. The column efficiencies were determined by examining the resolution of the 271/281 base pair doublet in the separation of a phiX174 HaeIII DNA sizing ladder. Tapered turns with the smallest radius of curvature (250 microm), the shortest tapering length between the separation and turn widths (55 microm), and the largest tapering ratio (4:1 separation channel width to turn channel width) produced the highest resolution separations. These results are discussed by comparison to theoretical predictions of the effect of tapers and turns on analyte band dispersion in capillary electrophoresis.