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.     

Correlation of capillary zone electrophoresis with continuous free-flow zone electrophoresis: application to the analysis and purification of synthetic growth hormone releasing peptide

Two carrier-free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free-flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purifity of peptide fractions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correlation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow-through cell, respectively, have to be taken into account and corresponding corrections have to be made.     

INTERVAL CARRIER FREE ELECTROPHORESIS FOR HIGH RESOLUTION PROTEIN PURIFICATION

Interval free flow zone electrophoresis is a new mode of free flow zone electrophoresis (FFZE), which facilitates purification of proteins and other molecular substances at very high resolution. It can be performed in the commercially available free flow electrophoresis (FFE) apparatus Octopus. The specimens are loaded and unloaded as usual with the help of a thin buffer film flowing between the two glass plates of the FFE. However, as long as electrical current is applied to a specimen, the medium flow is turned off and conditions of static column electrophoresis are simulated within the FFE device. Thereby electrohydrodynamic flow effects, which widen the sample bands migrating within the electric field, are eliminated while optimal heat removal from the thin buffer film is still possible and a sophisticated technique of harvesting of fractions remains available. Thus interval FFZE offers a gentle preparative method for purification of many kinds of charged molecular species such as proteins or dyes at very high resolution.     

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.     

Comparison of antimicrobial peptide purification via free‐flow electrophoresis and gel filtration chromatography

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad‐spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free‐flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS‐PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30‐fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2‐fold dilution but the latter had ∼13‐fold dilution. Furthermore, Tricine‐SDS‐PAGE, Native‐PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.     

Miniaturizing free-flow electrophoresis - a critical review

Free-flow electrophoresis (FFE) separation methods have been developed and investigated for around 50 years and have been applied not only to many types of analytes for various biomedical applications, but also for the separation of inorganic and organic substances. Its continuous sample preparation and mild separation conditions make it also interesting for online monitoring and detection applications. Since 1994 several microfluidic, miniaturized FFE devices were developed and experimentally characterized. In contrast to their large-scale counterparts microfluidic FFE (mu-FFE) devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the microliter range. Eventually, these mu-FFE systems might find application in so-called lab-on-a-chip devices for real-time monitoring and separation applications. This review gives detailed information on the results so far published on mu-FFE chips, comprising its four main modes, namely free-flow zone electrophoresis (FFZE), free-flow IEF (FFIEF), free-flow ITP (FFITP), and free-flow field-step electrophoresis (FFFSE). The principles of the different FFE modes and the basic underlying theory are given and discussed with special emphasis on miniaturization. Different designs as well as fabrication methods and applied materials are discussed and evaluated. Furthermore, the separation results shown indicate that similar separation quality with respect to conventional FFE systems, as defined by the resolution and peak capacity, can be achieved with mu-FFE separations when applying much lower electrical voltages. Furthermore, innovations still occur and several approaches for hyphenated, more integrated systems have been proposed so far, some of which are discussed here. This review is intended as an introduction and early compendium for research and development within this field.     

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra‐small detection cell length

Narrow peaks are important to high‐resolution and high‐speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra‐small detection cell length of 0.36 μm was easily achieved by using an image sensor.     

Protein separation using free‐flow electrophoresis microchip etched in a single step

A one‐step etching method was developed to fabricate glass free‐flow electrophoresis microchips with a rectangle separation microchamber (42 mm‐long, 23 mm‐wide and 28 μm‐deep), in which two glass bridges (0.5 mm‐wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free‐flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β‐lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na₂SO₄ added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free‐flow electrophoresis microchip fabricated by one‐step etching method is suitable for the continuous separation of proteins, which might become an effective pre‐fractionation method for proteome study.     

Study of two-dimensional liquid chromatography with high temperature NPLC and room temperature RPLC

To overcome the peak band broadening and to increase the peak capacity and separation efficiency of a two-dimensional liquid chromatographic system, a high-temperature normal phase liquid chromatography (HTNPLC) was used as the first dimension (1^st-D), and a RPLC was used as the second dimension (2^nd-D). The sample was first separated on the 1^st-D CN column and the primary eluent stored in the sampling-loop system alternatively (in HTNPLC×RPLC mode) or selectively (in HTNPLC/RPLC mode) and was then transferred to 2^nd-D C₁₈ column for further separation. The resolution and separation efficiency of the systems were greatly improved. The systems were evaluated by analyzing several polycyclic aromatic hydrocarbons and Glycyrrhiza uralensis extract. __________ Translated from Journal of Instrumental Analysis, 2008, 27(1) (in Chinese)     

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.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Two dimensional separations of human urinary protein digest using a droplet-interfaced platform

For highly complex mixtures, coelution is a common phenomenon in chromatography. A great deal of resolution is hidden in coelution, and lost due to inevitable molecular diffusion during sample transfer. The molecular diffusion may lead to band broadening and remix of separated peaks, which cause degradation of achievable resolution. In this study, we introduced droplet microfluidics as a high performance sample transfer tool in two dimensional nanoflow liquid chromatography–capillary electrophoresis separation of a human urine sample. The fine fractionation capability and sampling completeness enabled by the droplet-interface demonstrated the 2D system’s usefulness in high-resolution mapping of real world biological samples.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

New peak broadening parameter for the characterization of separation capability in capillary electrophoresis

The influence of separation conditions on peak broadening is usually estimated by the number of theoretical plates. Using the data available in literature and experimental data, it is shown that in pressure‐assisted capillary electrophoresis the plate number is not directly related to the separation capability of conditions used. The experiments at different electrolyte flow velocities demonstrate that a higher plate number (the best separation efficiency) can be obtained with a lower peak resolution. Since ions are separated by electrophoresis due to the difference in electrophoretic mobilities, the peak width in terms of electrophoretic mobility is suggested as a new peak broadening parameter describing the separation capability of the conditions used. The parameter can be calculated using the tailing factor and the temporal peak width at 5% of the peak height. A simple equation for the resolution calculation is derived using the parameter. The advantage of the peak width in terms of mobility over other parameters is shown. The new parameter is recommended to be used not only in pressure‐assisted capillary electrophoresis but also in general capillary electrophoresis when in a number of runs the virtual separative migration distance and separation capability of the conditions used change widely.     

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.     

Use of a heterogeneous buffer combination in microchip electrophoresis for high-resolution separation by on-line concentration of DNA samples

We have developed a novel high-resolution separation technique of DNA fragments in a heterogeneous combination of a sample buffer and a separation buffer. The use of a heterogeneous buffer combination is a simple method for on-line concentration of DNA fragments, in which a sample buffer is simply exchanged with one including taurine anions. The mobility of taurine anions, co-ions for DNA, is lower than the that of acetate anions in a separation buffer. The difference in the mobility invokes transient isotachophoresis. The current technique allows DNA fragments to be effectively concentrated and the separation length of microchips to be shorter than that of conventional ones by a factor of three without deterioration in separation resolution and any modification of a chip design. Fragments of 100-bp DNA ladders (100-1000 bp) were separated with high resolution (0.72-10.7) within 60 s with a 10 mm separation length on a polymethyl methacrylate chip. Furthermore, fragments of 10-bp DNA ladders (10-330 bp) were separated with high resolution (0.69-2.00) with a 10 mm separation length within 50 s without band broadening. The current achievements will make it possible to fabricate compact devices for microchip electrophoresis.     

Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA

Single-molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single-molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs 27 kbp) and small DNA (100 vs 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles (～150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.     

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.