Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.     

Preparative-scale isoelectric trapping enantiomer separations

The new Gradiflow BF200 IET unit, developed for isoelectric trapping protein separations has been modified and used to carry out preparative-scale enantiomer separations. Hydroxypropyl beta-cyclodextrin was used as the chiral resolving agent to induce an isoelectric point difference between the enantiomers. Three isoelectric membranes with isoelectric points below, in between and above the isoelectric points of the complexed enantiomers were used to trap the separated enantiomers in the anodic and cathodic separation compartments of the Gradiflow BF200 IET apparatus, respectively. The production rates were about 15 times higher than those previously obtained with another isoelectric trapping device and about 30% higher than those obtained in a continuous free-flow electrophoretic device operated in the isoelectric focusing mode. The remarkable separation speed observed in the modified Gradiflow BF200 IET unit is attributed to the favorable interplay of the short electrophoretic transfer distance, the high electric field strength and the large effective surface areas of the isoelectric membranes.     

Use of full-column imaging capillary isoelectric focusing for the rapid determination of the operating conditions in the preparative-scale continuous free-flow isoelectric focusing separation of enantiomers

A rapid, simple method is proposed here for the identification of the experimental conditions that lead to satisfactory preparative-scale isoelectric focusing enantiomer separations in continuous free-flow electrophoretic units. The method first calls for the use of a commercially available, full-column imaging capillary electrophoretic system to find the background electrolyte composition that generates the largest pI difference between the bands of the enantiomers. The method then calls for the finding of the minimum residence time that permits full development of the pH gradient across the separation chamber of the continuous free-flow electrophoretic unit by measuring the pH in the sample-free carrier electrolyte fractions collected during these runs. Finally, the quality of the predicted preparative-scale separation is verified by analyzing the enantiomer-containing collected fractions by capillary electrophoresis using a 14-sulfated, single-isomer cyclodextrin as resolving agent. The pI difference values and production rate values observed in this work agree well with the literature values that were obtained by much more time-consuming methods.     

Use of single-isomer, multiply charge chiral resolving agents for the continuous, preparative-scale electrophoretic separation of enantiomers based on the principle of equal-but-opposite analyte mobilities

A novel approach to continuous, preparative-scale electrophoretic enantiomer separations has been developed based on the observation that stable, equal-but-opposite effective mobilities can be created for the enantiomers of a single-charged analyte by complexing them with a single-isomer, multiply charged resolving agent, provided that the charge of the resolving agent is opposite in sign to that of the uncomplexed analyte enantiomers. When such an analyte-resolving agent system is fed into a continuous, free-flow electrophoretic apparatus, stable, steady-state operating conditions can be established which permit the continuous feeding of the racemic analyte and the collection of pure enantiomers at the opposite sides of the feed stream. This concept is demonstrated via the separation of the enantiomers of terbutaline using heptakis-6-sulfato beta-cyclodextrin as resolving agent, affording production rates as high as 2.8 mg/h in the general-purpose, continuous free-flow electrophoretic system, the Octopus.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

Increasing the scale of true moving bed electrophoretic separations using filtration to reduce solvent volumetric flows between sections II and III

Over the past decade the moving bed process has become a commonly used tool for the continuous separation of chiral compounds, and its recent application to electrophoretic separations allows the technique to be used as a model system for moving bed method improvements. Much of the recent research on moving bed separations has focused on improving the technique's efficiency and increasing the maximum attainable throughput. This paper presents a novel method for reducing or reversing the increases in tailing that stem from the addition of the feed stream in a moving bed process by adding a filtration unit which retains the products while removing fluid from the boundary between the sections above and below the feed stream. This filtration-enhanced moving bed process was applied to a true moving bed (TMB) electrophoresis separation in the Vortex Stabilized Electrophoresis Apparatus, and its effect on a homatropine enantiomer separation was studied. Experiments showed that there is a 2.4-fold increase in the homatropine processing rate when 0.5 ml/h of water is removed through a reverse osmosis filter at the boundary between the sections above and below the feed stream. In order to further understand the process, filtration-enhanced TMB (FE-TMB) was also analyzed using a linear model of the system which shows that the 99% purity operating region of the separation is greatly increased even with moderate permeate flowrates.     

Fundamental aspects of chiral separations by capillary electrophoresis

A review is presented that surveys the basic theory of direct separation of enantiomers by capillary electrophoretic (CE) techniques. These separations are based on the formation of diastereomeric complexes between the enantiomeric analytes and a chiral selector added to the electrolyte solution. The review covers a comprehensive treatment of the equations needed for optimization of selectivity coefficients, resolution and analysis time in the zone electrophoretic mode. In this context, it takes into account combined equilibria of complexation and protonation/deprotonation as well as complexation and paritition into micelles. On the basis of these equations, the benefits of charged selectors and the optimization potential inherent to pH tuning can be documented. In addition, the review deals with some basic aspects of chiral isoelectric focusing and briefly discusses indirect enantioseparation. In a subsequent section a survey is given on particularfeatures of the various types of chiral selectors. Finally, the recent developments in preparative enantioseparation in continuous free-flow system and by use of isoelectric membranes are discussed.     

Controlling of band width,resolution and sample loading by injection system in a simple preparative free-flow electrophoresis with gratis gravity

In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity.     

Isoelectric focusing field-flow fractionation : III. Investigation of the influence of different experimental parameters on focusing of cytochrome c in the trapezoidal cross-section channel

In addition to the electric field and pH gradient used in isoelectric focusing, a recently introduced technique, isoelectric focusing (or electrical hyperlayer) field-flow fractionation, employs the flow of the liquid carrier through a thin separation channel as a third factor affecting separation. Focusing of cytochrome c (CYTC) in a trapezoidal cross-section channel of 0.875 ml volume and 25 cm length was investigated as a function of the injection procedure, relaxation time, flow-rate of the carrier ampholyte solution and applied electric power. The influence of different initial conditions was also investigated by computer simulation. Both computed and experimental data showed an important contribution of the injection procedure and relaxation time on the retention and shape of the CYTC zone. It follows from these data that the sample should be injected as a narrow zone into the centre of the stream rather than homogeneously together with the carrier solution. For the described experimental set-up it could be demonstrated that the time necessary for zone formation should be at least 15 min and that relaxation times in excess to 20 min do not influence the final shape of the CYTC zone. It could further be shown experimentally that the sample must be injected under an applied electric field, that the relaxation time should be about 10 min, that the elution flow-rate should not be larger than 100 μl/min, that focusing becomes more efficient with increasing electric fields and that, for a given assembly and specified flow conditions, there is an electric power window only within which proper operation is possible.     

Determination of cobalt in urine by gas chromatography-mass spectrometry employing nickel as an internal standard.

In addition to the electric field and pH gradient used in isoelectric focusing, a recently introduced technique, isoelectric focusing (or electrical hyperlayer) field-flow fractionation, employs the flow of the liquid carrier through a thin separation channel as a third factor affecting separation. Focusing of cytochrome c (CYTC) in a trapezoidal cross-section channel of 0.875 ml volume and 25 cm length was investigated as a function of the injection procedure, relaxation time, flow-rate of the carrier ampholyte solution and applied electric power. The influence of different initial conditions was also investigated by computer simulation. Both computed and experimental data showed an important contribution of the injection procedure and relaxation time on the retention and shape of the CYTC zone. It follows from these data that the sample should be injected as a narrow zone into the centre of the stream rather than homogeneously together with the carrier solution. For the described experimental set-up it could be demonstrated that the time necessary for zone formation should be at least 15 min and that relaxation times in excess to 20 min do not influence the final shape of the CYTC zone. It could further be shown experimentally that the sample must be injected under an applied electric field, that the relaxation time should be about 10 min, that the elution flow-rate should not be larger than 100 μl/min, that focusing becomes more efficient with increasing electric fields and that, for a given assembly and specified flow conditions, there is an electric power window only within which proper operation is possible.     

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique—isoelectric focusing of proteins and a single-stranded DNA fragment

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm⁻¹ and 422 V cm⁻¹, depending on the application.     

Improved preparative-scale. continuous, free-flow electrophoretic separation of the enantiomers of terbutaline utilizing equal-but-opposite enantiomer mobilities.

The factors that influence yield and product purity in the continuous, preparative-scale electrophoretic separation of the enantiomers of terbutaline when using the principle of equal-but-opposite effective mobilities were studied. The sodium salt of heptakis-6-sulfato-beta-cyclodextrin was used as the resolving agent, in acidic, isopropanol-containing background electrolytes, in the continuous, free-flow, preparative electrophoretic instrument, the Octopus. By matching the linear velocity of the feed solution to that of the background electrolyte, lateral hydrodynamic dispersion was minimized resulting in a nonelectrophoresed feed band that was only three fractions (about 3 mm) wide as it exited the 0.5 m long separation channel. The multiple of residence time and applied potential was also optimized, constrained by migration of the front of heptakis-6-sulfato-beta-cyclodextrin out of the separation zone, leading to the recovery of 95% of both enantiomers in better than 99.99% purity, at a production rate of 0.1 mg/h.     

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78–4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.     

The Biflow: an instrument for transfer-loop mediated, continuous, preparative-scale isoelectric trapping separations

The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4 相似文献   

Experimental determination of sample stream focusing with fluorescent dye

This study aims to develop an experimental method for determining the focused-sample stream width in microfluidic chips. The focused-sample stream width in the junction of a cross-linked microchannel is determined by the distance between the two streamlines dividing the focusing sheath streams and the sample stream, which should be insensitive to the sample properties. However, the commonly used FWHM intensity method is dependent on the dye properties and cannot be used to estimate the sample focusing effects very accurately. The proposed method is based upon the fact that the cross-stream concentration profile of the sample stream in a region downstream of the junction matches the Gaussian distribution. Therefore, the width of the focused sample stream can be estimated from the channel widths and the variance and maximum concentration of the experimentally measured concentration profile. To validate this method, fluorescein dye has been used to obtain the concentration profile. It has been shown that the proposed method is independent of the fluorescent dye properties. Comparison between the proposed method, FWHM method, and some reported analytical methods also validates the proposed method.     

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.     

Two-dimensional gel isoelectric focusing

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

Unsteady transport phenomena in free-flow electrophoresis--prerequisite of ultrafast sample cleaning in microfluidic devices

The evolution partial differential equations describing the transport processes induced by hydrodynamic flow in free-flow electrophoresis (FFE) are solved by the generalized dispersion theory. Our theoretical analysis demonstrates that the central injection of solutes into a relatively fast hydrodynamic flow enables to transport them to the channel outlet well before they are spread through the width of the channel and their migration is negatively affected by a contact with walls. In this case, the axial zone spreading decreases by increasing the linear velocity of hydrodynamic flow. The resulting dependencies of convective and dispersion coefficients on the velocity of flow and parameters of the separation channel show the optimum separation conditions with respect to resolution and analysis time. Due to the unsteady character of transport processes, effective FFE separations can potentially be performed in a microfluidic device in seconds. This is a reasonable time to separate low-molecular mass impurities in the electric field. Thus, a fast and efficient sample cleaning before subsequent analysis by electrospray ionization-mass spectrometry (ESI-MS) or another separation method can be performed.