Biomedical subjects

Evan Shave

Publications and source records attributed to Evan Shave.

8 recordsLinked to original sources

PubMed2007

pH transients during salt removal in isoelectric trapping separations: a curse revisited.

The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH 7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (mu(anion)/mu(cation)) and the pH difference between pH 7 and the pH of the buffering membranes (|pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|). When |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1, mu(anion)/mu(cation)<1 leads to an acidic pH transient, mu(anion)/mu(cation) = 1 eliminates the pH transient and mu(anion)/mu(cation)>1 leads to a basic pH transient. When mu(anion)/mu(cation) = 1, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|<1 leads to a basic pH transient, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1 eliminates the pH transient and |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| value, pH transients caused by dissimilar anion and cation mobilities can be avoided.

Animals↗

PubMed2006

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations.

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3<pI<10 range, were prepared by blending. The master blend served as the feed mixture in binary isoelectric trapping separations that used the buffering membrane to be characterized. The pI value of the most basic UV-absorbing or fluorescent carrier ampholyte collected in the anodic separation compartment, determined by full-column imaging capillary isoelectric focusing analysis, indicates the operational pH value of the separation membrane.

Acrylates↗

PubMed2004

Preparative-scale isoelectric trapping separations in methanol-water mixtures.

The typically low aqueous solubilities of small, hydrophobic organic ampholytic molecules limit the production rates that can be achieved in their isoelectric trapping (IET) separations and call for the use of hydro-organic mixtures as solvents. The compatibility of methanol-water mixtures and poly(ethylene terephthalate) substrate-supported isoelectric polyacrylamide hydrogels, developed for binary IET separations in a Gradiflow BF200IET unit, was investigated. The isoelectric polyacrylamide-based hydrogels retained their functional and mechanical integrities when the methanol concentration in the hydro-organic solvent mixture was kept at or below 25% (v/v). The utility of the hydro-organic media was demonstrated in the purification of a hydrophobic ampholytic compound, technical grade 4-hydroxy-3-(morpholinomethyl) benzoic acid. Production rates as high as 7 mg/h were achieved using small, 15 cm2 active surface area isoelectric membranes.

Isoelectric Focusing↗

PubMed2004

Preparative-scale, recirculating, pH-biased binary isoelectric trapping separations.

In order to improve the production rates and lower the specific electrophoretic energy consumption values in preparative-scale, recirculating, binary isoelectric trapping separations, we propose to add an auxiliary isoelectric agent to the solution in the anodic separation compartment and another to the solution in the cathodic separation compartment to implement pH-biased isoelectric trapping. The auxiliary isoelectric agents are selected such that they are trapped in the respective anodic and cathodic separation compartments and also, have isoelectric point (pI) values that are different from the pI values of the analytes of interest. By proper selection of the auxiliary isoelectric agents and their concentrations, the analytes of interest can be kept in nonisoelectric, charged state during the entire course of the preparative-scale, recirculating, binary isoelectric trapping separation. This results in higher electrophoretic mobilities and solubilities for the analytes than in their isoelectric or near-isoelectric states, and leads to faster binary isoelectric trapping separations.

Animals↗

PubMed2004

Alkali-stable high-pI isoelectric membranes for isoelectric trapping separations.

Alkali-stable, high-pI isoelectric membranes have been synthesized from quaternary ammonium derivatives of cyclodextrins and poly(vinyl alcohol), and bifunctional cross-linkers, such as glycerol-1,3-diglycidyl ether. The new, high-pI isoelectric membranes were successfully applied as cathodic membranes in isoelectric trapping separations in place of the hydrolytically more labile, polyacrylamide-based cathodic isoelectric membranes, and permitted the use of catholytes as alkaline as 1 M NaOH. The new high-pI isoelectric membranes have shown excellent mechanical stability, low electric resistance and long life times, even when subjected to electrophoresis with current densities as high as 80 mA/cm2.

Cross-Linking Reagents↗

PubMed2004

High-buffering capacity, hydrolytically stable, low-pI isoelectric membranes for isoelectric trapping separations.

Hydrolytically stable, low-pI isoelectric membranes have been synthesized from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. The acidic functional groups are selected such that the pI value of the ampholytic component is determined by the pK(a) values of the acidic functional groups. When the concentration of the ampholytic component incorporated into the membrane is higher than a required minimum value, the pI of the membrane becomes independent of variations in the actual incorporation rate of the ampholytic compound. The new, low-pI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH < 1.5 anolytes and replaced the hydrolytically less stable polyacrylamide-based isoelectric membranes. The new low-pI isoelectric membranes have excellent mechanical stability, low electric resistance, good buffering capacity, and long life time, even when used with as much as 50 W power and current densities as high as 33 mA/cm(2) during the isoelectric trapping separations.

Buffers↗

PubMed2003

Preparative-scale isoelectric trapping enantiomer separations.

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

Isoelectric Focusing↗

PubMed2002

Preparative-scale isoelectric trapping separations using a modified Gradiflow unit.

The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.5 isoelectric membrane. The desalting and subsequent binary separation steps proved to be remarkably rapid, due to the favorable combination of short electrophoretic path, high electric field strength and large effective isoelectric membrane surface area.

Isoelectric Focusing↗