Negative‐pressure‐induced collector for a self‐balance free‐flow electrophoresis device

Uneven flow in free‐flow electrophoresis (FFE) with a gravity‐induced fraction collector caused by air bubbles in outlets and/or imbalance of the surface tension of collecting tubes would result in a poor separation. To solve these issues, this work describes a novel collector for FFE. The collector is composed of a self‐balance unit, multisoft pipe flow controller, fraction collector, and vacuum pump. A negative pressure induced continuous air flow rapidly flowed through the self‐balance unit, taking the background electrolyte and samples into the fraction collector. The developed collector has the following advantages: (i) supplying a stable and harmonious hydrodynamic environment in the separation chamber for FFE separation, (ii) effectively preventing background electrolyte and sample flow‐back at the outlet of the chamber and improving the resolution, (iii) increasing the preparative scale of the separation, and (iv) simplifying the operation. In addition, the cost of the FFE device was reduced without using a multichannel peristaltic pump for sample collection. Finally, comparative FFE experiments on dyes, proteins, and cells were carried out. It is evident that the new developed collector could overcome the problems inherent in the previous gravity‐induced self‐balance collector.     

Purification of low‐concentration phenazine‐1‐carboxylic acid from fermentation broth of Pseudomonas sp. M18 via free flow electrophoresis with gratis gravity

The low‐concentration phenazine‐1‐carboxylic acid (PCA) (=0.3 mM) extracted from fermentation broth of Pseudomonas sp. M18 was selected to be purified with a newly facile free flow electrophoresis (FFE) device with gratis gravity. Three factors of pH value and concentration of background buffer, and the cooling circle of FFE device were investigated for the purification of PCA in the FFE device. It was found that the pH value and concentration of background buffer had mild influences on the separation of PCA whether with cooling circle or not. However, the cooling circle had a much greater impact on the separation of PCA. The controlling of the band zone of PCA in FFE chamber would be difficult if without cooling circle, while the controlling would become easy if with cooling circle. Under the optimal conditions (10 mM pH 5.5 phosphate as background buffer, 30 mM pH 5.5 phosphate buffer as electrode solution, 5.46 mL/min background flux, 10 min residence time of injected sample, and 500 V), PCA could be continuously prepared from its impurities with relative high purity. The flux of sample injection was 115 μL/min, viz. 7 mL sample throughput per hour, and the recovery was up to 85%. All of the experiments indicated that the FFE technique was a good alternative tool for the study on natural biological control agents.     

Simply enhancing throughput of free‐flow electrophoresis via organic‐aqueous environment for purification of weak polarity solute of phenazine‐1‐carboxylic acid in fermentation of Pseudomonas sp. M18

Herein, a simple novel free‐flow electrophoresis (FFE) method was developed via introduction of organic solvent into the electrolyte system, increasing the solute solubility and throughput of the sample. As a proof of concept, phenazine‐1‐carboxylic acid (PCA) from Pseudomonas sp. M18 was selected as a model solute for the demonstration on feasibility of novel FFE method on account of its faint solubility in aqueous circumstance. In the developed method, the organic solvent was added into not only the sample buffer to improve the solubility of the solute, but also the background buffer to construct a uniform aqueous‐organic circumstance. These factors of organic solvent percentage and types as well as pH value of background buffer were investigated for the purification of PCA in the FFE device via CE. The experiments revealed that the percentage and the types of organic solvent exerted major influence on the purification of PCA. Under the optimized conditions (30 mM phosphate buffer in 60:40 (v/v) water‐methanol at an apparent pH 7.0, 3.26 mL/min background flux, 10‐min residence time of injected sample, and 400 V), PCA could be continuously purified from its impurities. The flux of sample injection was 10.05 μL/min, and the recovery was up to 93.7%. An 11.9‐fold improvement of throughput was found with a carrier buffer containing 40% (v/v) methanol, compared with the pure aqueous phase. The developed procedure is of evident significance for the purification of weak polarity solute via FFE.     

Preparative free‐flow electrophoretic offline ESI‐Fourier transform ion cyclotron resonance/MS analysis of Suwannee River fulvic acid

Free‐flow electrophoresis (FFE), a preparative free zone electrophoretic method, was used offline in conjunction with ultrahigh‐resolution FT/ion cyclotron resonance ‐MS to resolve the complexity of Suwannee River fulvic acid (SRFA). Before MS, the FFE separation conditions and the compatibility with ESI were optimized. The constituents in SRFA were effectively separated based on their charge states and sizes. The obtained mass spectra were compared by means of van Krevelen diagrams and the calculated aromaticity indices of the individual constituents were used to describe the distribution of aromatic/unsaturated structures across the FFE‐fractionated samples. The consolidated number of ions observed within the individual SRFA fractions were much higher than those of the bulk samples alone, demonstrating extensive ion suppression effects in bulk SRFA likely also operating in the analysis of complex biogeochemical mixtures in flow injection mode. The FFE approach allows for producing sizable amounts of sample from dilute solutions, which can be easily fractionated into dozens of individual samples with the possibility of further in‐depth characterization.     

Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.     

Enhancing resolution of free‐flow zone electrophoresis via a simple sheath‐flow sample injection

In this work, a simple and novel sheath‐flow sample injection method (SFSIM) is introduced to reduce the band broadening of free‐flow zone electrophoresis separation in newly developed self‐balance free‐flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C‐phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free‐flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath‐flow sample injection method.     

A microchip device to enhance free flow electrophoresis using controllable pinched sample injections

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.     

Chiral separation of phenylalanine and tryptophan by capillary electrophoresis using a mixture of β‐CD and chiral ionic liquid ([TBA] [l‐ASP]) as selectors

In this paper, a simple, effective and green capillary electrophoresis separation and detection method was developed for the quantification of underivatized amino acids (dl ‐phenylalanine; dl ‐tryptophan) using β‐Cyclodextrin and chiral ionic liquid ([TBA] [l ‐ASP]) as selectors. Separation parameters such as buffer concentrations, pH, β‐CD and chiral ionic liquid concentrations and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in a background electrolyte composed of 15 mm sodium tetraborate, 5 mm β‐CD and 4 mm chiral ionic liquid at pH 9.5, and an applied voltage of 10 kV. Under optimum conditions, linearity was achieved within concentration ranges from 0.08 to 10 µg/mL for the analytes with correlation coefficients from 0.9956 to 0.9998, and the analytes were separated in less than 6 min with efficiencies up to 970,000 plates/m. The proposed method was successfully applied to the determination of amino acid enantiomers in compound amino acids injections, such as 18AA‐I, 18AA‐II and 3AA. Copyright © 2013 John Wiley & Sons, Ltd.     

Micro free-flow electrophoresis: theory and applications

Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (μFFE) over the past 15 years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in μFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.

A simple chip free‐flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye

Here, a simple micro free‐flow electrophoresis (μFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο‐BBV) and fluorescent dye. The μFFE contained two open electrode cavities and an ion‐exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed μFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion‐exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 μm, 77 μL) avoiding the discard of μFFE due to blockage of solute precipitation in chamber. Remarkably, the μFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο‐BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10^?11 M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the μFFE.     

Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free-flow electrophoresis devices

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC–micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of < 30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20 min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.     

Size separation of graphene oxide using preparative free‐flow electrophoresis

Graphene oxide nanosheets often bear a wide size distribution. However, it is critical to have nanosheets with narrow size distribution for their unique size‐dependent physiochemical properties, and nanosheets with a narrow size distribution are the cornerstones for application. Therefore, efficient separation methods of graphene nanosheets have been given considerable attention in many scientific areas recently. Free‐flow electrophoresis is extensively used in the separation and purification of biological molecules with continuous flow separation. The charged graphene oxide nanosheets to some extent are very close in size to biological molecules and share similarity in motion behavior in an electric field. Thus, in the present work, we present a new and simple means to separate graphene oxide nanosheets into more mono‐dispersed size groups by using the free‐flow electrophoresis technique. By optimizing the separation conditions, we were able to obtain graphene oxide sheets with narrow size distribution. The separated samples were characterized by atomic force microscopy, and the size measurements were made by using the software “Image Pro Plus.” In addition, a brief discussion is also given into the theoretic background of the separation of graphene oxide according to the size by the technique of preparative free‐flow electrophoresis.     

Chip‐Based Free‐Flow Electrophoresis with Integrated Nanospray Mass‐Spectrometry

Free‐flow electrophoresis is an ideal tool for preparative separations in continuous microflow. With the approach presented herein for coupling free‐flow electrophoresis and mass spectrometry it is now also possible to trace non‐fluorescent compounds and identify them by means of mass spectrometry. The functionality of the method and its potential as an integrated separation unit for microflow synthesis is demonstrated by application to a multicomponent [3+2]‐cycloannulation.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

Quantitative investigation of resolution increase of free-flow electrophoresis via simple interval sample injection and separation

Interval free‐flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free‐flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10^?3). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two‐model antibiotics (herein pyoluteorin and phenazine‐1‐carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.     

Rapid separation of cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside from crude mulberry extract using high‐performance countercurrent chromatography and establishment of a volumetric scale‐up process

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside, using high‐performance countercurrent chromatography, and the establishment of a volumetric scale‐up process from semi‐preparative to preparative‐scale. To optimize the separation parameters, biphasic solvent systems composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi‐preparative‐scale high‐performance countercurrent chromatography. The optimized semi‐preparative‐scale high‐performance countercurrent chromatography parameters (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200–1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative‐scale (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0–10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside. The separation of mulberry anthocyanins using semi‐preparative high‐performance countercurrent chromatography and its volumetric scale‐up to preparative‐scale was addressed for the first time in this report.     

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

Free‐flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home‐made carrier ampholyte‐free FFIEF system was constructed via orientated migration of H⁺ and OH^? provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R² = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte‐free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI‐TOF‐MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris‐acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.     

Microfluidic preparative free‐flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free‐flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular‐shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion‐permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50×70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free‐flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule‐heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370±20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

Experimental study on the optimization of general conditions for a free‐flow electrophoresis device with a thermoelectric cooler†

With a given free‐flow electrophoresis device, reasonable conditions (electric field strength, carrier buffer conductivity, and flow rate) are crucial for an optimized separation. However, there has been no experimental study on how to choose reasonable general conditions for a free‐flow electrophoresis device with a thermoelectric cooler in view of Joule heat generation. Herein, comparative experiments were carried out to propose the selection procedure of general conditions in this study. The experimental results demonstrated that appropriate conditions were (i) <67 V/cm electric field strength; (ii) lower than 1.3 mS/cm carrier buffer conductivity (Tris‐HCl: 20 mM Tris was titrated by HCl to pH 8.0); and (iii) higher than 3.6 mL/min carrier buffer flow rate. Furthermore, under inappropriate conditions (e.g. 400 V voltage and 40 mM Tris‐HCl carrier buffer), the free‐flow electrophoresis separation would be destroyed by bubbles caused by more Joule heating. Additionally, a series of applications under the appropriate conditions were performed with samples of model dyes, proteins (bovine serum albumin, myoglobin, and cytochrome c), and cells (Escherichia coli, Streptococcus thermophilus, and Saccharomyces cerevisiae). The separation results showed that under the appropriate conditions, separation efficiency was obviously better than that in the previous experiments with randomly or empirically selected conditions.