Moving reaction boundary and isoelectric focusing: IV. Systemic study on Hjertén's pH gradient mobilization

In this paper, Hjertén's mobilization of pH gradient in IEF was systemically and quantitatively analyzed with the R_r value of judgment expression comparing the fluxes of hydrogen and hydroxyl ions. The theoretical results show that (i) there is R_r = 0, viz., quasi‐equal fluxes of proton and hydroxyl ion, in a classic IEF with sulfuric acid and sodium hydroxide used as the anolyte and catholyte, respectively, this is the main reason why pH gradient is quite stable in IEF; (ii) but if the salt of sodium sulfate is added into the sodium hydroxide, there is R_r > 0, viz., the flux of proton being higher than that of hydroxyl ion, the R_r value implies a cathodic mobilization of pH gradient, and the higher the R_r value is the faster the cathodic mobilization becomes; (iii) if the salt is added into the sulfuric acid, there is R_r < 0, the R_r value indicates an anodic mobilization, and the smaller the R_r value is the faster the anodic mobilization turns. To test these theoretical results above, a novel procedure was developed for the run of classic IEF followed by Hjertén's mobilization of pH gradient. The strict experiments were in well coincidence with the theoretical results. The results have obvious significances for the mechanism and development of Hjertén's mobilization.     

The effect of pH adjusted electrolytes on capillary isoelectric focusing assessed by high-resolution dynamic computer simulation

The effect of the composition of electrolytes on capillary IEF is assessed for systems with carrier ampholytes covering two pH units and with catholytes of decreased pH, anolytes of increased pH, and both electrode solutions with adjusted pH values. For electrolytes composed of formic acid as anolyte and ammonium hydroxide as catholyte, simulation is demonstrated to provide the expected IEF system in which analytes with pI values within the formed pH gradient are focused and become immobile. Addition of formic acid to the catholyte results in the formation of an isotachophoretic zone structure that migrates toward the cathode. With ammonium hydroxide added to the anolyte migration occurs toward the anode. In the two cases, all carrier components and amphoteric analytes migrate isotachophoretically as cations or anions, respectively. The data reveal that millimolar amounts of a counter ion are sufficient to convert an IEF pattern into an ITP system. With increasing amounts of the added counter ion, the overall length of the migrating zone structure shrinks, the range of the pH gradient changes, and the migration rate increases. The studied examples indicate that systems of this type reported in the literature should be classified as ITP and not IEF. When both electrolytes are titrated, a non-uniform background electrolyte composed of formic acid and ammonium hydroxide is established in which analytes migrate according to local pH and conductivity without forming IEF or ITP zone structures. Simulation data are in qualitative agreement with previously published experimental data.     

The transitional isoelectric focusing process

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.     

At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysis

Sample preparation is a crucial step for protein analysis. Functionalized magnetic nanoparticle (MNP)-based extraction has been developed to be a useful sample preparation technique for proteomic analysis. In this paper, we present a strategy for at-line coupling of MNP-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use an anolyte or a catholyte as the desorbing agent. Thus, functionalized MNPs can be facilely at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added to the sample well, which can function as a natural adapter. Once a focusing electric field has been applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as a desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals their pI values. The at-line combination was well demonstrated with three types of functionalized nanoparticles: (1) phenylboronic acid functionalized MNPs for extracting glycoproteins through boronate affinity; (2) carboxyl-functionalized MNPs for extracting positively charged proteins through a weak cation exchange mechanism; and (3) amino-functionalized MNPs for extracting negatively charged proteins through a weak anion exchange mechanism. The at-line combination exhibited several significant advantages, including selectivity, sensitivity, and speed.     

Investigation of the pH gradient formation and cathodic drift in microchip isoelectric focusing with imaged UV detection

This paper reports the protein analysis by using microchip IEF carried on an automated chip system. We herein focused on two important topics of microchip IEF, the pH gradient and cathodic drift. The computer simulation clarified that the EOF could delay the establishment of pH gradient and move the carrier ampholytes (CAs) to cathode, which probably caused a cathodic drift to happen. After focusing, the peak positions of components in a calibration kit with broad pI were plotted against their pI values to know the actual pH gradient in a microchannel varying time. It was found that the formed pH gradient was stable, not decayed after readily steady state, and migrated to cathode at a rate of 10.0 μm/s that determined by the experimental conditions such as chip material, internal surface coating and field strength. The theoretical pH gradient was parallel with the actual pH gradient, which was demonstrated in two types of microchip with different channel lengths. No compression of pH gradient was observed when 2% w/v hydroxypropyl methyl cellulose was added in sample and electrolytes. The effect of CAs concentration on current and cathodic drift was also explored. With the current automatic chip system, the calculated peak capacity was 23–48, and the minimal pI difference was 0.20–0.42 for the used single channel microchip with the effective length of 40.5 mm. The LOD for the analysis of CA‐I and CA‐II was around 0.32 μg/mL by using normal imaged UV detection, the detected amount is ca. 0.07 ng.     

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.     

Nonamphoteric isoelectric focusing: II. Stability of borate-glycerol pH gradients in recycling isoelectric focusing

By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0-6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.     

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.     

Application of Ion-Exchange Membranes for Stabilizing Acidity of a Chromium-Plating Electrolyte Based on Chromium(III) Sulfate

The acidity of a chromium-plating electrolyte based on chromic sulfate may be stabilized in a two-chamber electrolyzer with an anion-exchange membrane (AEM). The optimum concentration of sulfuric acid in the anolyte (c _sa) may be selected on the basis of the material balance equation for the catholyte, which allows for the dependence of the transport number of hydronium ions in AEM on c _sa. No considerable accumulation of Cr²⁺ occurs in the catholyte during a prolonged electrolysis. The transport of formate ions through AEM at working values of electrolyte pH is inconsiderable.     

Instabilities of the pH gradient in carrier ampholyte-based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation

Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte-based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte-based focusing in closed and open columns. Focusing under realistic conditions within a 5 cm long capillary in which three amphoteric low molecular mass dyes were focused in a pH 3–10 gradient formed by 140 carrier ampholytes was investigated. In open columns, electroosmosis displaces the entire gradient toward the cathode or anode whereas the electrophoretic processes act bidirectionally with a transition around pH 4 (drifts for pI > 4 and pI < 4 typically toward the cathode and anode, respectively). The data illustrate that focused zones of carrier ampholytes have an electrophoretic flux and that dynamic simulation can be effectively used to assess the magnitude of each of the electrokinetic destabilizing factors and the resulting drift for a combination of these effects. Predicted drifts of focused marker dyes are compared to those observed experimentally in a setup with coated capillary and whole column optical imaging.     

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.     

Parallel processing in the isoelectric focusing chip

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.     

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.     

Carrier ampholyte‐free isoelectric focusing on a paper‐based analytical device for the fractionation of proteins

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte‐free isoelectric focusing on paper‐based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry. This paper‐based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost‐effective protein sample clean‐up method for target protein analysis with mass spectrometry.     

Electrophoretic mobilization in capillary isoelectric focusing by a weak acid or an acidic ampholyte as catholyte assessed by computer simulation

Capillary isoelectric focusing (CIEF) with cationic electrophoretic mobilization induced via replacing the catholyte with the anolyte or a solution of another acid or amino acid was investigated by computer simulation for a wide range pH gradient bracketed between two amphoteric spacers and short electrode vials with a higher id than the capillary. Dynamic simulations provide insight into the complexity of the mobilizing process in a hitherto inaccessible way. The electrophoretic mobilizing process begins with the penetration of the mobilizing compound through the entire capillary, is followed by a gradual or steplike decrease of pH, and ends in an environment with a non-homogenous solution of the mobilizer. Analytes do not necessarily pass the point of detection in the order of decreasing pI values. Cationic mobilization encompasses an inherent zone dispersing and refocusing process toward the capillary end. This behavior is rather strong with phosphoric acid and citric acid, moderate with aspartic acid, glutamic acid (GLU), formic acid, and acetic acid and less pronounced in the absence of the cathodic spacer. The data reveal that optical detectors should not be placed before 90% of capillary length. Aspartic acid, GLU, formic acid, and acetic acid provide an environment with a continuously decreasing pH that explains their successful use in optimized two-step CIEF protocols.     

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.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Stable Operation of Aqueous Organic Redox Flow Batteries in Air Atmosphere

As a green route for large-scale energy storage, aqueous organic redox flow batteries (AORFBs) are attracting extensive attention. However, most of the reported AORFBs were operated in an inert atmosphere. Herein, we clarify this issue by using the reported AORFB (i.e., 3, 3′-(9,10-anthraquinone-diyl)bis(3-methylbutanoicacid) (DPivOHAQ)||Ferrocyanide) as an example. We demonstrate that the dissolved O₂ can oxidize the discharged DPivOHAQ in anolyte, leading to capacity-imbalance between anolyte and catholyte. Therefore, this cell shows continuous capacity fading when operated in an air atmosphere. We propose a simple strategy for this challenge, in which the oxygen evolution reaction (OER) in catholyte is employed to balance oxygen reduction reaction (ORR) in anolyte. When using the Ni(OH)₂-modifed carbon felt (CF) as a current collector for catholyte, this cell shows an excellent stability in air atmosphere because the Ni(OH)₂-induced OER capacity in catholyte exactly balances the ORR capacity in anolyte. Such O₂-balance strategy facilitates AORFBs’ practical application.