On-line Separations Research Articles

Coupling Online Size Exclusion Chromatography with Charge Detection-Mass Spectrometry Using Hadamard Transform Multiplexing.

Charge detection mass spectrometry (CD-MS) is a powerful technique for the analysis of large, heterogeneous biomolecules. By directly measuring the charge states of individual ions, CD-MS can measure the masses from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. However, CD-MS is inherently slow because hundreds or thousands of spectra need to be collected to produce adequate ion statistics. The slower speed of CD-MS complicates efforts to couple it with online separation techniques, which limit the number of spectra that can be acquired during a chromatographic peak. Here, we present the application of Hadamard transform multiplexing to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS, with a goal of using SEC for separating complex mixtures prior to CD-MS analysis. We developed a microcontroller to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. Data showed a series of peaks spaced according to the pseudorandom injection sequence, which were demultiplexed with a Hadamard transform algorithm. The demultiplexed data revealed improved CD-MS signals while preserving retention time information. This multiplexing approach provides a general solution to the inherent incompatibilities of online separations and CD-MS detection that will enable a range of applications.

Sample transformation in online separations: how chemical conversion advances analytical technology.

While the advent of modern analytical technology has allowed scientists to determine the complexity of mixtures, it also spurred the demand to understand these sophisticated mixtures better. Chemical transformation can be used to provide insights into properties of complex samples such as degradation pathways or molecular heterogeneity that are otherwise unaccessible. In this article, we explore how sample transformation is exploited across different application fields to empower analytical methods. Transformation mechanisms include molecular-weight reduction, controlled degradation, and derivatization. Both offline and online transformation methods have been explored. The covered studies show that sample transformation facilitates faster reactions (e.g. several hours to minutes), reduces sample complexity, unlocks new sample dimensions (e.g. functional groups), provides correlations between multiple sample dimensions, and improves detectability. The article highlights the state-of-the-art and future prospects, focusing in particular on the characterization of protein and nucleic-acid therapeutics, nanoparticles, synthetic polymers, and small molecules.

Charge Inversion of Mono- and Dianions to Cations via Triply Charged Metal Complexes: Application to Lipid Mixtures.

Electrospray ionization (ESI) of mixtures can give rise to ions with different masses and charges with overlapping mass-to-charge (m/z) ratios. Such a scenario can be particularly problematic for the detection of low-abundance species in the presence of more highly abundant mixture components. For example, negative mode ESI of polar lipid extracts can result in highly abundant singly charged glyerophospholipids (GPLs), such as phosphatidylethanolamines (PE) and phosphatidylglycerols (PG), that can obscure much less abundant cardiolipins (CLs), which are complex phospholipids with masses roughly double those of GPLs that mostly form doubly charged anions. Despite their low relative abundance, CLs are lipidome components that perform vital biological functions. To facilitate the study of CLs in lipid mixtures without resorting to offline or online separations, we have developed a gas-phase approach employing ion/ion reactions to charge invert anionic lipid species using a trivalent metal-complex. Specifically, ytterbium(III) is shown to readily complex with three neutral ligands, N,N,N',N'-tetra-2-ethylhexyl diglycolamide (TEHDGA), to form [Yb(TEHDGA3)]3+ using ESI. Herein, we describe pilot studies to evaluate [Yb(TEHDGA)3]3+ as an ion/ion reagent to allow for chemical separation of doubly and singly charged anions, using lipid mixtures as examples, without neutralizing ions of either charge state.

Automated Control of Injection Times for Unattended Acquisition of Multiplexed Individual Ion Mass Spectra.

Charge detection mass spectrometry (CDMS) provides mass domain spectra of large and highly heterogeneous analytes. Over the past few years, we have multiplexed CDMS on Orbitrap instruments, an approach termed Individual Ion Mass Spectrometry (I2MS). Until now, I2MS required manual adjustment of injection times to collect spectra in the individual ion regime. To increase sample adaptability, enable online separations, and reduce the barrier for entry, we report an automated method for adjusting ion injection times in I2MS for image current detectors like the Orbitrap. Automatic Ion Control (AIC) utilizes the density of signals in the m/z domain to adjust an ensemble of ions down to the individual ion regime in real-time. The AIC technique was applied to both denatured and native proteins yielding high quality data without human intervention directly in the mass domain.

Tuning of salt separation efficiency by flow rate control in microfluidic dynamic dialysis

Microliter-scale separation processes are important for biomedical research and point-of-care diagnostics with small-volume clinical samples. Analytical assays such as mass spectrometry and field effect sensing necessitate sample desalting, but too low a salt concentration can disrupt protein structures and biomolecular interactions. In this work, we investigated whether salt extraction from a protein solution can be controlled by dynamic dialysis parameters. A microfluidic counter-flow dialyzer with a 5 kDa molecular weight cut-off cellulose membrane was fabricated by laser cutting and operated with a wide range of feed and dialysis flow rates. It was found that with the appropriate flow conditions, most notably the feed flow rate, retentate salt concentrations from 0.1 to 99% of the input NaCl concentration can be achieved. The experimental data were in good agreement with a theoretical diffusion-based mass transfer model. The salt dialysis performance was similar in the presence of 50 mg/mL albumin, approximating blood plasma protein content, and did not deteriorate with overnight continuous dialysis, indicating minimal membrane fouling. The dialyzer construction method is compatible with all planar membranes, enabling implementation of tuneable dynamic dialysis for a wide range of on-line microfluidic biomolecular separations.

Top-down protein identification using a time-of-flight mass spectrometer and data independent acquisition

Top-down mass spectrometry and direct dissociation of gas phase intact proteins have been demonstrated to be a powerful platform for identifying proteins from complex mixtures and for elucidating post-translational modifications (PTMs). Fragmentation of proteins in the atmospheric pressure/vacuum interface of the electrospray ionization mass spectrometer is an effective dissociation technique that can be utilized for on-line HPLC top-down analysis. We demonstrate the capability to perform intact protein identifications in a single-stage time-of-flight (TOF) mass spectrometer in a data independent (DIA) acquisition fashion by rapidly switching the in-source dissociation (ISD) energy during protein elution from a liquid chromatography (LC) column. The intact protein and product ion masses obtained at low and high ISD energies, respectively, were measured using a TOF mass analyzer. By coupling on-line protein separations to dissociation in the atmospheric pressure/vacuum interface region of the mass spectrometer, we identified proteins in simple complexity mixtures, including subunits from the human 20S proteasome complex, and PTMs such as phosphorylation and N-terminal acetylation events. This proof-of-principle study demonstrates that a data-independent pseudo-MS/MS method could be a relatively inexpensive platform for top-down MS.

High Speed Intact Protein Characterization Using 4X Frequency Multiplication, Ion Trap Harmonization, and 21 Tesla FTICR-MS

Mass spectrometric characterization of large biomolecules, such as intact proteins, requires the specificity afforded by ultrahigh resolution mass measurements performed at both the intact mass and product ion levels. Although the performance of time-of-flight mass analyzers is steadily increasing, the choice of mass analyzer for large biomolecules (e.g., proteins >50 kDa) is generally limited to the Fourier transform family of mass analyzers such as Orbitrap and ion cyclotron resonance (FTICR-MS), with the latter providing unmatched mass resolving power and measurement accuracy. Yet, protein analyses using FTMS are largely hindered by the low acquisition rates of spectra with ultrahigh resolving power. Frequency multiple detection schemes enable FTICR-MS to overcome this fundamental barrier and achieve resolving powers and acquisition speeds 4× greater than the limits imposed by magnetic field strength. Here we expand upon earlier work on the implementation of this technique for biomolecular characterization. We report the coupling of 21T FTICR-MS, 4X frequency multiplication, ion trapping field harmonization technology, and spectral data processing methods to achieve unprecedented acquisition rates and resolving power in mass spectrometry of large intact proteins. Isotopically resolved spectra of multiply charged ubiquitin ions were acquired using detection periods as short as 12 ms. Large proteins such as apo-transferrin (MW = 78 kDa) and monoclonal antibody (MW = 150 kDa) were isotopically resolved with detection periods of 384 and 768 ms, respectively. These results illustrate the future capability of accurate characterization of large proteins on time scales compatible with online separations.

Parallel detection in a single ICR cell: Spectral averaging and improved S/N without increased acquisition time

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is well-renowned for its ultrahigh resolving power and mass measurement accuracy. As with other types of analytical instrumentation, achievable signal-to-noise ratio (S/N) is an important analytical figure of merit with FTICR-MS. S/N can be improved with higher magnetic fields and longer time-domain signal acquisition periods. However, serial signal averaging of spectra or time-domain signals acquired with multiple ion populations is most commonly used to improve S/N. On the other hand, serial acquisition and averaging of multiple scans significantly increases required data acquisition time and is often incompatible with on-line chromatographic separations. In this study, we investigated the potential for increased S/N by averaging 4 spectra that were acquired in parallel with a single ICR cell with 4 pairs of dipole detection electrodes, each with an independent pre-amplifier. This spectral averaging was achieved with no need for multiple ion accumulation events nor multiple, serial excitation and detection events. These efforts demonstrated that parallel signal acquisition with 4 detector electrode pairs produces S/N 1.76-fold higher than that from a single detection electrode pair. With parallel detection, improved S/N was achieved with no observable loss in resolving power (100,000) as compared with that from a single detection electrode pair. These results demonstrate that parallel detection of multiple induced image current signals with multiple preamplifiers exists as a viable option for future instrumentation to increase achievable S/N and sensitivity. This approach may have general utility especially where conventional serial signal averaging is impractical.

Comprehensive two dimensional liquid chromatography as analytical strategy for pharmaceutical analysis

Comprehensive on-line two-dimensional liquid chromatography (LCxLC) is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceuticals. A comparative study of different sets of chromatographic conditions including stationary phase, pH additive and organic modifier was carried out with two real pharmaceutical samples in order to find out the best analytical conditions for implementation of one or several generic on-line LCxLC separations. Our choice was based on the evaluation of both degree of orthogonality and practical sample peak capacity under linear gradient conditions. The potential of 190 combinations of chromatographic systems was compared. A set of 3 RPLCxRPLC configurations was found to be very attractive for both samples and in good agreement with the findings of a previous study carried out with 17 model compounds, thereby supporting the idea of using generic LCxLC conditions in the pharmaceutical area. The three selected 2D-systems were implemented for the on-line RPLCxRPLC-UV/MS analysis of two pharmaceutical samples. It was shown, for each sample, that these 2D-systems were able to generate an effective peak capacity close to 1000 in less than 50min. For each sample, baseline separation was obtained for every known compound and furthermore a large number of unknown impurities could also be separated and identified. Finally, in the proposed conditions, the total number of compounds detected was significantly improved from one RPLC separation to one RPLCxRPLC separation. Only a small additional gain was observed by performing a second RPLCxRPLC separation or even a third one.

Negative Electron Transfer Dissociation Sequencing of Increasingly Sulfated Glycosaminoglycan Oligosaccharides on an Orbitrap Mass Spectrometer.

The structural characterization of sulfated glycosaminoglycan (GAG) carbohydrates remains an important target for analytical chemists attributable to challenges introduced by the natural complexity of these mixtures and the defined need for molecular-level details to elucidate biological structure-function relationships. Tandem mass spectrometry has proven to be the most powerful technique for this purpose. Previously, electron detachment dissociation (EDD), in comparison to other methods of ion activation, has been shown to provide the largest number of useful cleavages for de novo sequencing of GAG oligosaccharides, but such experiments are restricted to Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS). Negative electron transfer dissociation (NETD) provides similar fragmentation results, and can be achieved on any mass spectrometry platform that is designed to accommodate ion-ion reactions. Here, we examine for the first time the effectiveness of NETD-Orbitrap mass spectrometry for the structural analysis of GAG oligosaccharides. Compounds ranging in size from tetrasaccharides to decasaccharides were dissociated by NETD, producing both glycosidic and cross-ring cleavages that enabled the location of sulfate modifications. The highly-sulfated, heparin-like synthetic GAG, ArixtraTM, was also successfully sequenced by NETD. In comparison to other efforts to sequence GAG chains without fully ionized sulfate constituents, the occurrence of sulfate loss peaks is minimized by judicious precursor ion selection. The results compare quite favorably to prior results with electron detachment dissociation (EDD). Significantly, the duty cycle of the NETD experiment is sufficiently short to make it an effective tool for on-line separations, presenting a straightforward path for selective, high-throughput analysis of GAG mixtures. Graphical Abstract ᅟ.

Fraction transfer process in on-line comprehensive two-dimensional liquid-phase separations.

Two-dimensional liquid-phase separations have gained increasing attention for their ability to separate complex sample mixtures. Among the experimental setups used, an on-line approach is preferred to reduce the probability of sample contamination, for easier automation and high-sample throughput. The interfacing of the separation techniques in the on-line mode brings additional demands on proper optimization of the two-dimensional system. In this review, the possibilities of the on-line coupling of liquid chromatography and liquid chromatography with capillary electrophoresis in two-dimensional systems are discussed. Special attention is paid to the fraction transfer process, which includes an overview of interfaces and experimental setups applied, the compatibility issues of separation systems, and instrumental parameters. The benefits and drawbacks of using electromigration separations in combination with liquid chromatography are presented as well.

Potential and limitations of on-line comprehensive reversed phase liquid chromatography × supercritical fluid chromatography for the separation of neutral compounds: An approach to separate an aqueous extract of bio-oil

On-line comprehensive Reversed Phase Liquid Chromatography×Supercritical Fluid Chromatography (RPLCxSFC) was investigated for the separation of complex samples of neutral compounds. The presented approach aimed at overcoming the constraints involved by such a coupling. The search for suitable conditions (stationary phases, injection solvent, injection volume, design of interface) are discussed with a view of ensuring a good transfer of the compounds between both dimensions, thereby allowing high effective peak capacity in the second dimension. Instrumental aspects that are of prime importance in on-line 2D separations, were also tackled (dwell volume, extra column volume and detection). After extensive preliminary studies, an on-line RPLCxSFC separation of a bio-oil aqueous extract was carried out and compared to an on-line RPLCxRPLC separation of the same sample in terms of orthogonality, peak capacity and sensitivity. Both separations were achieved in 100min. For this sample and in these optimized conditions, it is shown that RPLCxSFC (with Hypercarb and Acquity BEH-2EP as stationary phases in first and second dimension respectively) can generate a slightly higher peak capacity than RPLCxRPLC (with Hypercarb and Acquity CSH phenyl-hexyl as stationary phases in first and second dimension respectively) (620 vs 560). Such a result is essentially due to the high degree of orthogonality between RPLC and SFC which may balance for lesser peak efficiency obtained with SFC as second dimension. Finally, even though current limitations in SFC instrumentation (i.e. large extra-column volume, large dwell volume, no ultra-high pressure) can be critical at the moment for on-line 2D-separations, RPLCxSFC appears to be a promising alternative to RPLCxRPLC for the separation of complex samples of neutral compounds.

Cr(VI) speciation in foods by HPLC-ICP-MS: investigation of Cr(VI)/food interactions by size exclusion and Cr(VI) determination and stability by ion-exchange on-line separations.

A method has been developed for the specific and sensitive determination of Cr(VI) in foods. First, the interactions between Cr(VI) and the matrices were investigated by size-exclusion HPLC-ICP-MS (SEC-ICP-MS). Evidence was found for the complexation of Cr(VI) potentially present with the ligands. Forquantification of Cr(VI), the method was based on an alkaline extraction (NH4OH solution at pH11.5) followed by Cr(VI) determination by anion-exchange HPLC-ICP-MS. Analytical performances of the method were satisfactory in terms of linearity, specificity, accuracy, repeatability, and intermediate precision. Detection limits ranged from 1 to 10μg/kg, depending on the matrices investigated. The method was then applied for the determination of Cr(VI) in several products (dairy products, flour, chocolate, vegetables, fruits, meat, fish, eggs, and beverages) from different brands and origins. Cr(VI) was found in none of the samples investigated. To further investigate the reason for this absence, a stability study of spiked Cr(VI) was therefore conducted. A semi-skimmed cow milk was selected for this study. Cr(VI) was shown to be unstable in this matrix with a degradation rate increasing with the temperature.

A simulation study of linear RF ion guides for AMS

The use of radiofrequency multipoles and particularly the radiofrequency quadrupole (RFQ) controlled gas cell to facilitate on-line isobar separations for Accelerator Mass Spectrometry (AMS) is being explored experimentally and theoretically in a preliminary way at present. These new methods have the potential to extend greatly the analytical scope of AMS. However, there are many technical challenges to adapt an RF gas cell isobar separating device and still maintain stable and high transmission for routine AMS using the high current Cs+ sputter ion sources developed for nuclear physics and adapted to the detection of rare radioactive isotopes for AMS. An overview of linear RF ion guide properties is therefore needed to assist in the conceptualization of their efficient additions into AMS. In this work the intrinsic properties of linear RF ion guides, which are relevant to the generation of the RF induced ion energy distributions and for the evaluation of the ion transmissions in vacuum, are systematically studied using SIMION 8.1. These properties are compared among radiofrequency quadrupole, hexapole and octupole ion guides, so that their usefulness for AMS applications can be evaluated and compared. By simulation it is found that to prepare a typical RF captured AMS ion beam to within a safe range of ion energies prior to the onset of gas interactions, a higher multipole is more suitable for the first RF field receptor, while a quadrupole operated with q2∼0.5 is more suited as the final ion guide for concentrating the energy-cooled ions near axis.

The One Hour Yeast Proteome

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: Orthogonality and practical peak capacity considerations

Comprehensive on-line two-dimensional liquid chromatography is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceutical or biological ones. A comparative study of different sets of chromatographic conditions including stationary phase, mobile phase and column temperature was carried out with mixtures of representative solutes in order to find out the best two-dimensional analytical conditions for charged compounds. Our approach focused on ultra-fast gradient runs in second dimension using HT-UHPLC conditions. The choice of volatile buffers was intended for future coupling with mass spectrometry in order to get another relevant dimension. The potential of various pairs of chromatographic systems was examined by means of two-dimensional gradient data. An attempt is made to rationalize the concept of orthogonality and a method is proposed to assess, for a given pair of chromatographic systems, both the degree of orthogonality and the practical peak capacity. It is shown that the degree of orthogonality between both dimensions is a critical factor but it is not sufficient to definitely appreciate the potential of a given pair of systems. The combination of HILIC and RPLC (HILIC×RPLC or RPLC×HILIC), although providing a very high degree of orthogonality, is disappointing due to the poor peak capacities obtained in HILIC especially with peptide samples. RPLC systems offer a large variety of analytical conditions, some of them leading to appropriate degrees of orthogonality when they are combined. More importantly, due to high column efficiencies along with large separation power, some combinations of RPLC systems leads to very high practical peak capacities.

Nuclear structure and reaction studies near doubly magic 270Hs

Abstract Fast on-line gas chemical separations of Hs (hassium, element 108) in the form of HsO4 were applied to investigate the reactions 26Mg + 248Cm and 36S + 238U. In an experiment at the gas-filled separator DGFRS the reaction 48Ca + 226Ra was studied. In all cases the product of complete nuclear fusion is 274Hs*. For the first time, the new nuclide 270Hs was produced in the 4n evaporation channel and its decay properties investigated. The nuclide 270Hs was predicted by microscopic-macroscopic calculations to be a deformed doubly magic nucleus and its decay properties are therefore of special interest to theory. Also, much more detailed information was gained on the decay of 269Hs and its daughters, which led to a new assignment of decay properties of the daughter nuclides 265Sg and 261Rf. There is evidence fo r isomeric states in 265Sg and 261Rf, while 266Sg is not an alpha-particle emitter as believed previously, but decays by spontaneous fission (SF) with a rather short half-life. Also, interesting features of the used reaction 26Mg + 248Cm led to the discovery of the nucleus 271Hs in the same experiments. An investigation of the influence of the Q-value on the fusion reaction in relation to the location of the fusion barrier showed, that the high binding energy of 48Ca largely compensates for the lower fusion probability compared to more asymmetric reactions, while 36S is not as promising as a projectile.

Relationship between selectivity and average resolution in comprehensive two-dimensional separations with spectroscopic detection

The average value of the multivariate selectivity ( SEL) of randomly positioned peaks in a multi-component separation is shown to equal the average fraction of peaks that are singlets, as predicted by statistical-overlap theory (SOT). This equality is the basis for proposing a useful metric, specifically the average minimum resolution of nearest-neighbor peaks, for the performance of comprehensive two-dimensional (2D) separations. Furthermore this metric was computed both without ancillary spectroscopic information and with the assistance of such help, specifically multi-wavelength UV–vis spectra, acquired during the separation. Separations are simulated with randomly positioned peaks over wide ranges of total number of peaks, first- and second-dimension peak capacity, dimensionless first-dimension sampling time, and spectral diversity. The specific version of the general multivariate selectivity concept that is used here – identified as SEL – gives the relative precision of quantification when using the PARAFAC (parallel factor analysis) method, a popular curve resolution algorithm. The SEL values of all peaks were calculated, averaged, and compared to the predictions of SOT. In the absence of auxiliary spectral data, the SEL-based average minimum resolution required to separate two peaks in a 2D separation is 0.256, compared to resolution of 0.5 if no chemometric assistance is available. This was found to be valid over a wide range of conditions and is essentially independent of peak crowding. With the assistance of the spectral data, the requisite minimum resolution substantially improves, that is, it decreases, especially when peak crowding is severe. The requisite minimum resolution decreases even further, up to a limit, as the spectral diversity is increased. In contrast, the SEL-based average under-sampling correction factor is virtually independent of the presence of the additional spectral data, and additionally is about the same as calculated with SOT from the average number of maxima in closely analogous simulations. The use of selectivity greatly increases the fraction of peaks that are singlets, relative to the number of singlet maxima, especially when spectral assistance is added. The insensitivity of the under-sampling correction factor to either the use of selectivity or added spectral data simplifies optimization of the corrected peak capacity in on-line comprehensive 2D separations.

Theoretical description of a new analytical technique: Comprehensive online multidimensional fast Fourier transform separations

Comprehensive multidimensional separations are today dominated by systems that are fundamentally limited to highly asymmetrical online separations sacrificing separation space, or to lengthy, time consuming offline separations. With the exception of pulse-modulated methods, separations have thus been limited to two dimensions. It is proposed that some of the limitations and shortcomings of these methods may be ameliorated or overcome by employing multi-dimensional detection whereby each analyte is effectively labelled in the frequency domain by a series of pulsed-injections, and a symmetrical, comprehensive online analysis performed with the resulting signal processed by sequential Fourier analysis. A semi-empirical computer model of this system was developed and its feasibility positively demonstrated in simulations of high-efficiency separations in two dimensions. Separations of higher dimensionality were shown to be possible but involved signal-processing challenges beyond the present work. By eliminating wrap-around effects and enabling the separation of physically unseparated peaks, the technique facilitates significant improvements in peak capacity per unit of analysis time as well as greatly improved signal to noise ratios. Because these comprehensive online multidimensional Fourier transform separations depend heavily upon the practical lifetime of imposed injection pulses, it is envisaged that this method will leverage emerging high-efficiency micro- and nanoscale separations technologies.

A theoretical and experimental study of the electrophoretic extraction of ions from a pressure driven flow in a microfluidic device

The electrophoretic extraction of ions from a hydrodynamic flow stream is investigated at an intersection between two microfluidic channels. A pressure gradient is used to drive samples through the main channel, while ions are electrophoretically extracted into the side channels. Hydrodynamic restrictors and a neutral coating are used to suppress bulk flow through the side channels. A theoretical model that assumes Poiseuille flow in the main channel and neglects molecular diffusion is used to calculate the extraction efficiency, eta, as a function of the ratio, R, of the average hydrodynamic velocity to the electrophoretic velocity. The model predicts complete extraction of ions (eta=1) for R<2/3 and a monotonic decrease in eta as R becomes greater than 2/3, which agrees well with the experimental results. Additionally, the model predicts that the aspect ratio of the microfluidic channel has little effect on the extraction efficiency. It is anticipated that this device can be used for on-line process monitoring, sample injection, and 2D separations for proteomics and other fields.