Electrokinetic sample transport in a microchannel with spatial electrical conductivity gradients

Studies of the sample transport in a microchannel with the electrical conductivity gradient are critical to develop techniques for on-chip sample transport control. A numerical model presented in this paper, consisting of the electrical potential equation, full Navier-Stokes equation and species conservation equation, is used to simulate sample transport in a microchannel with the consideration of the conductivity gradient. There are two situations studied here, sample pumping (where sample separation is minimized by employing a high-conductivity buffer in the sample region), and sample stacking (where sample separation is expedited by using a low-conductivity buffer as the sample carrier). The effects of applied electrical potential, sample diffusion coefficient and the ratio of conductivity of the driving buffer over the sample carrying buffer are investigated by using the developed model.     

Electrophoretic separations of neuromediators on microfluidic devices

In the present work, on-chip capillary electrophoresis for the separation of neuromediators is demonstrated. The influence of separation buffer (composition, pH, SDS additive), on-chip electrokinetic sample stacking, and surface pretreatment of the PDMS-PDMS and hybrid PDMS-glass devices on the electrokinetic characteristics of microfluidics (ν_eo, μ_eo, ζ) and separation performance of on-chip capillary electrophoresis of neuromediators have been investigated. It is demonstrated that for the effective separation of neuropeptides on elastomer-based microfluidic devices, on-chip sample stacking is necessary. Field-amplified sample stacking for electroosmotic flow supported on-chip separations of neuromediators and without special design of the sample injection scheme has been demonstrated. Electrophoretic separations of fluorescently labeled analytes have been achieved within tens of seconds at injection volumes of about 110 pL, with plate numbers varying from <1000 to ∼22,000. These results demonstrate that on-chip separation methods with hybrid PDMS-glass devices are perspective for the analysis of (neuro)peptides in small volumes.     

pH-independent large-volume sample stacking of positive or negative analytes in capillary electrophoresis

In capillary electrophoresis, the short optical path length associated with on-column UV detection imposes an inherent detection problem. Detection limits can be improved using sample stacking. Recently, large-volume sample stacking (LVSS) without polarity switching was demonstrated to improve detection limits of charged analytes by more than 100-fold. However, this technique requires suppression of the electroosmotic flow (EOF) during the run. This necessitates working at a low pH, which limits using pH to optimize selectivity. We demonstrate that LVSS can be performed at any buffer pH (4.0-10.0) if the zwitterionic surfactant Rewoteric AM CAS U is used to suppress the EOF. Sensitivity enhancements of up to 85-fold are achieved with migration time, corrected area, and peak height reproducibility of 0.8-1.6%, 1.3-3.7%, and 0.8-4.9%, respectively. Further, it is possible to stack either positively or negatively charged analytes using zwitterionic surfactants to suppress the EOF.     

Simultaneous enrichment and separation of neutral and anionic analytes through combining large volume sample stacking with sweeping in CE

In this work, we overcame the deficiencies of large volume sample stacking (LVSS) in separating low‐mobility and neutral analytes through combining LVSS with sweeping in CE, and employed this new approach to enrich and separate neutral and anionic analytes simultaneously. This technique was carried out with pressure injection of large‐volume sample followed by EOF as a pump pushing the bulk of low‐conductivity sample matrix out of the outlet of the capillary while analytes were swept by micelles and separated via MEKC without the electrode polarity switching. Careful optimization of the enrichment and separation conditions allowed the enrichment factors (EFs) of peak height and peak area of the analytes to be in the range of 9–33 and 21–35 comparing with the conventional injection mode, respectively. The five analytes were baseline separated in 15 min and the detection limits ranged from 26.5 to 55.8 ng/mL (S/N = 3). The developed method was successfully applied to determine adenine, caffeine, theophylline, reduced L‐glutathione (GSH) and oxidized L‐glutathione (GSSG) in two different teas with recoveries that ranged from 84.4 to 105.2%.     

Electric field gradient focusing

Electric field gradient focusing (EFGF) is a relatively new separation technique with promising attributes, particularly for protein analysis. The fundamental fractionation mechanism in EFGF involves a gradient in electric field along the length of a separation column. The electrophoretic force that drives charged analytes in a region of changing electric field is opposed by a constant, pressure-driven bulk fluid flow. When the electrophoretic velocity of a particular moiety is equal and opposite to the velocity of the fluid flow, the analyte focuses into a stationary band. Thus, EFGF can both concentrate and separate charged species according to electrophoretic mobility. To date, the electric field gradients needed for EFGF have been established using a number of different approaches, including channels having changing cross-sectional areas, conductivity gradients caused by the diffusion of buffer ions across a membrane, electrode arrays, and temperature gradients in buffers whose conductivities change as a function of temperature. EFGF has proven particularly effective for sample enrichment, with concentration factors of 10,000 reported. In this article we review advances in EFGF technology and discuss prospects for further improving EFGF for chemical analysis.     

Separation and online preconcentration by multistep stacking with large-volume injection of anabolic steroids by capillary electrokinetic chromatography using charged cyclodextrins and UV-absorption detection

The separation of three common anabolic steroids (methyltestosterone, methandrostenolone and testosterone) was performed for the first time by capillary EKC. Different charged CD derivatives and bile salts were tested as dispersed phases in order to achieve the separation. A mixture of 10 mmol/L succinylated-beta-CD with 1 mmol/L beta-CD in a 50 mmol/L borate buffer (pH 9) enabled the separation of the three anabolic steroids in less than 9 min. Concentration LODs, obtained for these compounds with low absorption of UV light, were approximately 5 x 10(-5) mol/L. The use of online reverse migrating sample stacking with large-volume injection (the effective length of the capillary) enabled to improve the detection sensitivity. Sensitivity enhancement factors (SEFs) ranging from 95 (for testosterone) to 149 (for methyltestosterone) were achieved by single stacking preconcentration. Then, the possibilities of multistep stacking to improve the sensitivity for these analytes were investigated. SEFs obtained by double stacking preconcentration ranged from 138 to 185, enabling concentration LODs of 2.79 x 10(-7) mol/L (for methyltestosterone), 3.47 x 10(-7) mol/L (for testosterone) and 3.56 x 10(-7) mol/L (for methandrostenolone). Although online triple stacking preconcentration was achieved, its repeatability was very poor and SEFs for the studied analytes were not calculated.     

Review of recent developments of on-line sample stacking techniques and their application in capillary electrophoresis

Capillary electrophoresis (CE) has become one of the most useful tools in separation science because of its high separation efficiency, low cost, versatility, ease of sample preparation and automation. However, some limitations of CE, such as poor concentration sensitivity due to its lower sample loading and shorter optical path length, limits its further applications in separation science. In order to solve this problem, various on-line sample preconcentration techniques such as transient isotachophoresis preconcentration, field-enhanced sample stacking, micelle to solvent stacking, micelle collapse, dynamic pH junction, sweeping, solid phase extraction, single drop microextraction and liquid phase microextraction have been combined with CE. Recent developments, applications and some variants together with different combinations of these techniques integrating in CE are reviewed here and our discussions will be confined to the past three years (2008–2011).      

Analyte zone sharpening in pressurized capillary electrochromatography based on electrophoretic migration under a heterogeneous field

Significant peak width reductions, or peak height enhancements, of angiotensins were observed when a high voltage was applied to hydrophilic interaction pressurized capillary electrochromatography using gradient elution with mobile phases containing perchloric acid. The investigation using a contactless conductivity detector revealed that perchloric acid was adsorbed on the surface of the stationary phase, when the acetonitrile content in the mobile phase was high, and released from the stationary phase by increasing the water content during a gradient procedure. The released perchloric acid formed a highly concentrated zone moving from the column inlet to the outlet. The electrochromatographic behavior of the analytes, primarily electrophoretic migration, was changed in this zone. As a consequence of the significant variation in migration velocity of the analytes, the sample band width was reduced similar fashion to on‐capillary concentration in capillary electrophoresis. Using this result, the reduction of band width and enhancement in separation efficiency was demonstrated in reversed‐phase pressurized electrochromatography, in which the conductivity of the mobile phase was significantly altered using a step gradient. The resolution between benzoic acid and 1‐naphthalene sulfonic acid was successfully improved from 2.7 to 4.3 by using the band width reduction method based on field‐amplified stacking.     

On‐line synergistic stacking in capillary zone electrophoresis featuring field‐amplified sample stacking and micelle to cyclodextrin stacking in the determination of two alkaloids in complicated matrix samples

A novel on‐line synergistic proconcentration strategy coupling field‐amplified sample stacking and micelle to cyclodextrin stacking for cationic analytes in capillary zone electrophoresis has been proposed and applied for the separation and determination of two alkaloids, matrine, and oxymatrine in complicated matrix samples. The approach was performed by the long injection of sample in a low‐conductivity sodium dodecyl benzene sulfonate solution followed by the injection of hydroxypropyl‐β‐cyclodextrin solution in higher conductivity. The stacking mechanism of this method has been expounded and parameters affecting stacking effect have been optimized in our study. Under the optimum experimental conditions, 169‐ and 218‐fold sensitivity improvements were achieved for matrine and oxymatrine when compared with normal injection. Analytical indicators including linearity, limits of detection, and reproducibility (intra‐ and inter‐day relative standard deviations) were evaluated. Moreover, sample matrix effect was studied using compound flavescent sophora and salicylic acid powder and spiked urine samples. The developed method is an attempt for the combination of micelle to cyclodextrin stacking with other stacking methods. It could be a good alternative choice for the determination of alkaloids in a complex sample matrix.     

Large-volume sample on-line concentration of angiotensins in non-aqueous capillary electrophoresis

A single step on-line concentration and separation method for peptides in non-aqueous capillary electrophoresis was developed. ACN containing 50 mM tetraethylammonium perchlorate was used as the electrophoretic medium; angiotensins I-IV were separated as a result of the differences in the magnitudes of their interactions with perchlorate anions. When the sample solution (ACN containing 0.5% trifluoroacetic acid and angiotensins) was injected as a large-volume plug, the analytes were concentrated at the inlet end of the capillary by both sweeping and stacking mechanisms; the separation procedure then started automatically without any operations such as polarity change. It was found that the concentration of analytes, injection period, and concentration of tetraethylammonium perchlorate in the electrophoretic medium were important factors for both separation and concentration efficiencies. The angiotensins were concentrated and separated with the large-volume injection of up to 80% of the effective capillary length.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

Sample self-stacking in capillary zone electrophoresis: behavior of samples containing multiple major coionic components

It is a frequent phenomenon in practice that a sample contains bulk levels of more than one coionic component that affect the stacking behavior of minor analytes and in this way also the sensitivity of the method. Here, attention is paid to stacking resulting from the presence of a macrocomponent of leading type that is deteriorated by the presence of another macrocomponent of like charge in the sample. Based on the isotachophoretic model of migration in the initial period of separation, a theoretical approach was elaborated both for strong and weak electrolytes which describes the separation process and finds the conditions that define whether transient isotachophoretic stacking of the analyte takes place or not. It is shown that the crucial parameter is the ratio of the concentrations of macrocomponents migrating in front and behind the analyte of interest. The destacking effect can also be expected when the coion of the background electrolyte is present in the sample. Rules how to cope with effects of destackers present in the sample are given. Theoretical considerations are illustrated by computer simulations and verified experimentally. Examples of antagonistic effects of macrocomponents are demonstrated for model serum samples.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Diffusion as major source of band broadening in field-amplified sample stacking under negligible electroosmotic flow velocity conditions

Theory of field-amplified sample stacking (FASS) also called field-enhanced sample stacking is reevaluated considering the early work of Chien, Burgi and Helmer. The classical theory presented by Chien, Helmer and Burgi predicts the existence of maxima, which are ascribed to the counteracting principles of zone focusing and hydrodynamic dispersion. In contrast to their work, we here focus on cationic analytes separated in an acidic background electrolyte providing a very low electroosmotic flow velocity. Therefore, peak broadening due to differences in the local electroosmotic flow velocities in different compartments of the capillary can be regarded to be negligible. Consequently, peak broadening resulting from hydrodynamic dispersion will not be the dominant limitation of the accessible enrichment efficiency. In our experimental studies we, however, obtain an optimum value for the field enhancement factor (maximum of the enrichment efficiency, when varying the electric conductivity of the sample and the size of the sample injection plug) corresponding to a 10-fold dilution of the BGE in the sample solution. Comparing these experimental data with data modeled according to the revised theory, we show that this limitation of the loadability is caused by the unavoidable decrease of the analyte migration velocity in the BGE compartment of the capillary when injecting of a sample plug of lower electric conductivity (decrease in the local electric field strength). The additional diffusional band broadening limits the obtainable enrichment efficiency.     

Investigation of the mechanism of pH-mediated stacking of anions for the analysis of physiological samples by capillary electrophoresis

Capillary electrophoresis has been widely used for the analysis of physiological samples such as plasma and microdialysate. However, sample destacking can occur during the analysis of these high-ionic strength samples, resulting in poor separation efficiency and reduced sensitivity. A technique termed pH-mediated stacking of anions (base stacking) has previously been developed to analyze microdialysate samples and achieve on-line preconcentration of analytes by following sample injection with an injection of sodium hydroxide. In this work, the mechanism of base stacking was investigated. Peak efficiency was shown to be a function of background electrolyte and sample ionic strength. Analytes representing several classes of compounds with a wide range of mobilities were used to study the effects of multiple parameters on sample stacking. The length of hydroxide injection required for stacking was shown to be dependent on analyte mobility and the type of amine background electrolyte used. Combinations of electrokinetic and hydrodynamic injections of sample and hydroxide were examined and it was concluded that although stacking could be achieved with several injection modes, electrokinetic injection of both sample and hydroxide was most effective for sample stacking. The mechanism of pH-mediated stacking for each of these modes is presented.     

Monosubstituted positively charged cyclodextrins: Synthesis and applications in chiral separation

Several series of novel structurally well-defined positively charged CDs, applicable to alpha-, beta- and gamma-CDs for chiral separation using CE and chromatographic techniques have been developed. The chiral resolution capabilities of different series CDs towards amino acids and anionic analytes in CE are systematically investigated by considering all separation parameters including CD type, alkyl chain length of the cations attached to the CD rim, CD concentration, buffer pH, separation temperature and organic solvent. Typical results are demonstrated in the context. Examples of chiral separation with HPLC and supercritical fluid chromatography are first demonstrated by using coated chiral stationary phases (CSPs). Optimum CD loading content on the coated CSPs was explored in the chiral separation of neutral analytes.     

On-chip microfluidic buffer swap of biological samples in-line with downstream dielectrophoresis

Microfluidic cell enrichment by dielectrophoresis, based on biophysical and electrophysiology phenotypes, requires that cells be resuspended from their physiological media into a lower conductivity buffer for enhancing force fields and enabling the dielectric contrast needed for separation. To ensure that sensitive cells are not subject to centrifugation for resuspension and spend minimal time outside of their culture media, we present an on-chip microfluidic strategy for swapping cells into media tailored for dielectrophoresis. This strategy transfers cells from physiological media into a 100-fold lower conductivity media by using tangential flows of low media conductivity at 200-fold higher flow rate versus sample flow to promote ion diffusion over the length of a straight channel architecture that maintains laminarity of the flow-focused sample and minimizes cell dispersion across streamlines. Serpentine channels are used downstream from the flow-focusing region to modulate hydrodynamic resistance of the central sample outlet versus flanking outlets that remove excess buffer, so that cell streamlines are collected in the exchanged buffer with minimal dilution in cell numbers and at flow rates that support dielectrophoresis. We envision integration of this on-chip sample preparation platform prior to or post-dielectrophoresis, in-line with on-chip monitoring of the outlet sample for metrics of media conductivity, cell velocity, cell viability, cell position, and collected cell numbers, so that the cell flow rate and streamlines can be tailored for enabling dielectrophoretic separations from heterogeneous samples.     

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10(-8) mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10(-3) mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.     

Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole--time-of-flight mass spectrometer

The application of microfabricated devices coupled to a quadrupole time-of-flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub-nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several microL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post-separation channel. Rapid identification of trace level peptides is also demonstrated using on-line tandem mass spectrometry and database searching with peptide sequence tags.