Biomedical subjects

Evan Shave

Publications and source records attributed to Evan Shave.

6 recordsLinked to original sources

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↗