Evolution of the theoretical description of the isoelectric focusing experiment: II. An open system isoelectric focusing experiment is a transient, bidirectional isotachophoretic experiment.

The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off.

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady-state model to the current two-stage model of isoelectric focusing.

In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss.

Evolution of the theoretical description of the isoelectric focusing experiment: III. Carrier ampholyte behavior in transient, bidirectional isotachophoresis.

In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( / values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, 675-88) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on.

Multi-cationic aminopyrene-based labeling tags for oligosaccharide analysis by capillary electrophoresis-mass spectrometry.

In this work, new multi-cationic aminopyrene-based labeling tags were designed and synthesized for oligosaccharide analysis by capillary electrophoresis-mass spectrometry (CE-MS). The starting compound, 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt, was modified in order to form a sulfonamide derivative having three tertiary amines in the label structure. The sulfonamide derivative was further methylated to generate three permanently charged quaternary ammonium moieties on the label.

Determination of binding constants of multiple charged cyclodextrin complexes by ACE using uncorrected and ionic strength corrected actual mobilities of the species involved.

In this study, the apparent binding constants and limiting mobilities of the multiply charged complexes of the Δ- and Λ-enantiomers of Ru(II)- and Fe(II)-polypyridyl associates ([Ru(2,2'-bipyridine) ] , [Ru(1,10-phenanthroline) ] , and [Fe(1,10-phenanthroline) ] ) with single-isomer 2,3-diacetylated-6-sulfated-cyclodextrins (CDs) (12Ac-6S-α-CD, 14Ac-7S-β-CD, and 16Ac-8S-γ-CD) were determined by ACE using uncorrected and ionic strength corrected actual mobilities of the species involved. Two limiting models were tested for the ionic strength correction of the actual mobilities based on an empirical relation for the ionic strength correction of multivalent ionic species. In model 1, the nominal values of the charge numbers (z ) and analytical concentrations (c ) of the above CD selectors in the BGEs were applied for calculation of the BGE ionic strength, as usual.

CE determination of the thermodynamic pK values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation.

Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpK = pKa - pKa < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.

Equivalent peak resolution: characterization of the extent of separation for two components based on their relative peak overlap.

Although the classical formula of peak resolution was derived to characterize the extent of separation only for Gaussian peaks of equal areas, it is often used even when the peaks follow non-Gaussian distributions and/or have unequal areas. This practice can result in misleading information about the extent of separation in terms of the severity of peak overlap. We propose here the use of the equivalent peak resolution value, a term based on relative peak overlap, to characterize the extent of separation that had been achieved.

Overcoming the aggregation problem: a new type of fluorescent ligand for ConA-based glucose sensing.

Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution.

Simulation of the effects of complex-formation equilibria in electrophoresis: III. Simultaneous effects of chiral selector concentration and background electrolyte pH.

This paper describes the results of the second-level testing of the simulation program Simul 5 Complex. We compare the published experimental results with the simulated migration behavior of the enantiomers at different pH and chiral selector concentration values and use the same optimization object function, separation selectivity, as the original papers. Simul 5 Complex proved to be a suitable tool for the prediction of the effective mobilities, separation selectivities, and migration order reversals in these pH-dependent and CD concentration dependent enantiomer separations.

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 2--operation in cascade mode.

A parallel multicompartmental electrolyzer recently developed for preparative-scale isoelectric trapping separations, trapping by recursive electrophoresis in a compartmentalized system, was set up to operate as a cascade of binary separations to produce at least one pure target ampholyte (or more, with additional separation heads) without other ampholytes ever entering (even transiently) the harvest stream. This mode of operation avoids the need for exhaustive electrophoresis and the accompanying long separation times brought about by the exponentially decreasing concentrations over the course of batch separations. Continuous operation can be achieved in the cascade mode by continuously feeding the sample into the first separation head configured with three flow-through compartments and continuously harvesting one (or more) target components in additional separation heads configured with two flow-through compartments, attached to the respective branching points.

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 1--construction and standard operation.

The performance of the current preparative-scale isoelectric trapping systems is limited by the serial arrangement of the separation compartments. A new system has been developed that achieves trapping by recursive electrophoresis in a compartmentalized system (T-RECS). T-RECS features (i) parallel-connected elementary separation heads with independent electrode compartments, feed compartments, and harvest compartments, (ii) orthogonal primary pH gradients and secondary pH gradients, (iii) directionally controlled convective analyte transport along the primary (resolving) pH gradients, and (iv) electrophoretic analyte transport along the secondary (harvesting) pH gradients.

Synthesis, analytical characterization and initial capillary electrophoretic use in an acidic background electrolyte of a new, single-isomer chiral resolving agent: heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β-cyclodextrin.

The sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)cyclomaltoheptaose (HAMS), the first single-isomer sulfated β-CD that carries the sulfo group exclusively at the C2 position, has been synthesized. The purity of each synthetic intermediate and of the final product was determined by hydrophilic interaction (HILIC) and reversed-phase HPLC. The structural identity of each intermediate and of the final product was verified by 1-D and 2-D NMR spectroscopy and MALDI-TOF MS.

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis.

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers.

Comparison of charge state distribution in commercially available sulfated cyclodextrins used as chiral resolving agents in capillary electrophoresis.

The charge state distributions of randomly sulfated cyclodextrins from Sigma-Aldrich and Beckman-Coulter, as well as single isomer sulfated cyclodextrins from TM Chemicals LP were investigated using hydrophilic interaction liquid chromatography (HILIC). A cross-linked diol phase and an unbonded silica phase were used as HILIC stationary phases. Groups of sulfated cyclodextrins with different charge states were resolved from each other, while regioisomers in a charge group were partially separated.

Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 2.

A micropreparative-scale multicompartmental electrolyzer called ConFrac has been developed and tested for isoelectric trapping separations. ConFrac can be operated in pass-by-pass mode or recirculating mode, using either asymmetrical feeding (feed enters only the anodic or the cathodic flow-through compartment) or symmetrical feeding (feed enters both the anodic and the cathodic flow-through compartment). Symmetrical feeding results in higher processing rates and is the preferred operation mode.

Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 1.

A multicompartmental electrolyzer called ConFrac has been developed and tested for micropreparative-scale isoelectric trapping separations. ConFrac contains n separate, minimalistic isoelectric trapping core units, each with a separate anode compartment, anodic flow-through compartment, collection compartment, cathodic flow-through compartment and a shared cathode compartment. The collection compartments are all isolated from each other and have volumes of 100 μL each.

Efficient electrophoretic method to remove neutral additives from protein solutions followed by mass spectrometry analysis.

A mass spectrometry (MS)-compatible, isoelectric point-based separation method for removal of neutral additives from protein solutions is described. The separation is performed by electrophoretic migration and trapping using a device referred to as membrane separated wells for isoelectric focusing and trapping (MSWIFT). Electrophoretic separation in the MSWIFT device is fast; the entire process can be carried out in a matter of minutes, and it does not require further sample cleanup prior to MS analysis.

Combining isoelectric point-based fractionation, liquid chromatography and mass spectrometry to improve peptide detection and protein identification.

The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry-compatible isoelectric trapping device that provides isoelectric point (pI)-based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes.

A systematic study in CIEF: defining and optimizing experimental parameters critical to method reproducibility and robustness.

A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10 View Article and Find Full Text PDF

Simulation of desalting that occurs during isoelectric trapping separations.

Simul 5, the simulation program based on the 1-D model of electrophoresis has been extended to simulate isoelectric trapping (IET) separations that take place in recirculating multicompartmental electrolyzers (MCEs). In the extended Simul 5, the simulated separation space between the anode and cathode can be divided into multiple segments to represent the anode compartment, separation compartment(s) and the cathode compartment. The compartments may have identical or different cross sections.

PVA-based tunable buffering membranes for isoelectric trapping separations.

PVA-based buffering membranes with tunable pH values were prepared on a PVA substrate by reacting PVA, glycerol-1,3-diglycidyl ether, -NH2 group-containing buffers and -NH2 group-containing titrants in the presence of sodium hydroxide. The pH of the buffering membranes could be tuned in the 3 View Article and Find Full Text PDF

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture.

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3 View Article and Find Full Text PDF

Synthesis of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic beta-CD and its use for enantiomer separations by CE.

The dichloride salt of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose (PEMEDA-BCD), the first single-isomer, monosubstituted, permanently dicationic beta-CD has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of analytes in acidic and basic BGEs. When the concentration of PEMEDA-BCD was changed in the BGEs, the resulting effective mobilities of the analytes and the respective separation selectivities followed the predictions of the ionic strength-corrected charged resolving agent migration model. Good separation selectivities and favorable normalized EOF mobilities allowed for the rapid, efficient separation of the enantiomers of anionic, weak acid and nonionic analytes in the low- and/or high-pH BGEs.

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.

Rapid isoelectric trapping in a micropreparative-scale multicompartment electrolyzer.

A multicompartmental electrolyzer called membrane-separated wells for isoelectric focusing and trapping (MSWIFT) has been developed and tested for micropreparative-scale isoelectric trapping separations. In the MSWIFT, the length of the heat conduction path from the center of the compartments to the wall is less than 1 mm. The compartments are made from 99.