Biomedical subjects

Cornelius F Ivory

Publications and source records attributed to Cornelius F Ivory.

10 recordsLinked to original sources

PubMed2005

Multistage isoelectric focusing in a polymeric microfluidic chip.

This paper reports a protocol that improves the resolving power of isoelectric focusing (IEF) in a polymeric microfluidic chip. This method couples several stages of IEF in series by first focusing proteins in a straight channel using broad-range ampholytes and then refocusing segments of the first channel into secondary channels that branch from the first one at T-junctions. Experiments demonstrate that several fluorescent proteins that had focused within a segment of the straight channel in the first stage were refocused at significantly higher resolution due to the shallower pH gradient and higher electrical field gradient. Two variants of green fluorescent protein from the second-stage IEF fractionation were further separated in a third stage. Three stages of IEF were completed in less than 25 min at electric field strengths ranging from 50 to 214 V/cm.

Dimethylpolysiloxanes↗

PubMed2005

Microchannel protein separation by electric field gradient focusing.

A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel.

Dimethylpolysiloxanes↗

PubMed2005

Isoelectric focusing in a poly(dimethylsiloxane) microfluidic chip.

This paper reports the application of ampholyte-based isoelectric focusing in poly(dimethylsiloxane) (PDMS) using methylcellulose (MC) to reduce electroosmosis and peak drift. Although the characteristics of PDMS make it possible to fabricate microfluidic chips using soft lithography, unstable electroosmotic flow (EOF) and cathodic drift are significant problems when this medium is used. This paper demonstrates that EOF is greatly reduced in PDMS by applying a dynamic coat of MC to the channel walls and that higher concentrations of MC can be used to increase the viscosity of the electrode solutions in order to suppress pH gradient drift and reduce "compression"of the pH gradient. To illustrate the effect of MC on performance, several fluorescent proteins were focused in microchip channels 5 microm deep by 300 microm wide by 2 cm long in 3-10 min using broad-range ampholytes at electric field strengths ranging from 25 to 100 V/cm.

Ampholyte Mixtures↗

PubMed2004

Preparative free-flow electrofocusing in a vortex-stabilized annulus.

This paper describes the development of an annular chamber designed for preparative free-flow electrophoresis which can operate at voltages up to 20 kV, electric fields up to 65 kV/m, and power densities as high as 10 MW/m(3). This apparatus uses counter-rotating fluid vortices to stabilize the aqueous medium in the annulus against natural convection while improving heat and mass transfer over vortex-free Couette flow. The vortices are generated by rotating the inner surface of the annulus and, if the rotor and stator have complementary shapes, dispersion induced by electrokinetic and electrohydrodynamic flows can be mitigated even at the highest operating voltages. Following a brief overview of contemporary annular free-flow instrumentation, the theoretical principles for momentum and heat transport in the vortices are discussed in some detail and then the results of several electrofocusing experiments are provided to illustrate the resolving power of this instrument.

Electrophoresis↗

PubMed2004

Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus.

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.

Ampholyte Mixtures↗

PubMed2004

Peak compression and resolution for electrophoretic separations in diverging microchannels.

We report the results of experiments and simulations on electrokinetic flow in diverging microchannels (with cross-sectional area that increases with distance along the channel). Because of conservation of mass and charge, the velocity of an analyte in the channel decreases as the channel cross-section increases. Consequently, the leading edge of a band of sample moves more slowly than the trailing edge and the sample band is compressed. Sample peak widths, rather than increasing diffusively with time, can then be controlled by the geometry of the channel and can even be made to decrease with time. We consider the possibility of using this peak compression effect to improve the resolution of electrophoretic separations. Our results indicate that for typical separations that are dispersion limited, this peak compression effect is more than offset by the decreased distance between peaks, and the separation resolution in diverging channels is worse than that found for straight channels at the same applied voltage. For separations in very short channels or at very high field strengths, however, when the separation efficiency is injection limited, the peak compression effect is dominant and diverging channels can then be used to achieve improved separation resolution.

Electrophoresis, Microchip↗

PubMed2003

Microchip countercurrent electroseparation.

We report a microchip-based method for separating charged molecules according to electrophoretic mobility. The method is based on opposed electroosmotic, electrophoretic and convective forces. Similar to isoelectric focusing, solute can be accumulated into stationary zones, but without use of ampholytes. The method of "microchip countercurrent electroseparation" described here has potential application in the design of microfluidic separation chips.

Journal Article↗

PubMed2002

Continuous fractionation of enantiomer pairs in free solution using an electrophoretic analog of simulated moving bed chromatography.

Continuous fractionation of the left and right enantiomers of Piperoxan was performed in free solution in a vortex-stabilized electrophoresis apparatus. Sulfated beta-cyclodextrin was used as the chiral selector. A capillary electrophoresis (CE) study of the separation of Piperoxan enantiomers was carried out in order to find the buffer conditions that produce the maximum peak separation time between the two enantiomers and the optimal chiral selector concentration. These peak separation times were then used to calculate the electrophoretic mobilities of the enantiomer-ligand complexes. The difference in electrophoretic mobilities, when used in a preliminary model of the enantiomer separation, indicated that, by imposing a fluid flow opposite the direction of electromigration, it would be possible to force the fast and slow enantiomers to move in opposite directions within the vortex-stabilized apparatus. Using the predictions of the preliminary separation model, the vortex stabilized electrophoresis apparatus was configured with a feed port at the center of the chamber axis and offtake ports near the cathode and anode. This allowed for continuous operation of the apparatus. Continuous fractionations were completed at throughputs of 1.5 and 4.0 mg/h with both offtakes showing greater than 99% enantiomeric purity at 4.0 mg/h using CE. Fractionation was achieved at a throughput of 10 mg/h, but while the slow enantiomer was recovered with greater than 99% purity, only 96% enantiomeric purity of the fast stereoisomer was achieved. The loss of resolution at higher volumetric throughputs supports our hypothesis that a mobility-dependent "window" of operation exists in which two solutes can be completely separated.

Chromatography, Ion Exchange↗

PubMedDate not supplied

Modeling two-component isoelectric focusing buffers in a vortex-stabilized electrophoresis apparatus.

Defined buffer systems have been considered by researchers as replacements for synthetic carrier ampholyte mixtures in IEF in order to reduce both costs and deleterious protein-ampholyte interactions. In this paper, a mathematical model for the behavior of two-component pH gradients during IEF is applied to a vortex-stabilized electrophoresis apparatus. Equations for mass conservation, molar fluxes, and the electric field describe the behavior of the components in the electric field. Equilibrium constants are used in the model to account for interconversion between the positive, negative, and neutral states for each component. The model was applied to predict pH gradients using two different defined buffer systems. The model's predictions fell within the 95% confidence interval of a least-squares fit to the experimental pH gradients except in the regions near the ends of the pH gradients, where large pH excursions occurred during the experiments.

Ampholyte Mixtures↗

PubMedDate not supplied

Development of a segmented model for a continuous electrophoretic moving bed enantiomer separation.

With the recent demonstration of a continuous electrophoretic "moving bed" enantiomer separation at mg/h throughputs, interest has now turned to scaling up the process for use as a benchtop pharmaceutical production tool. To scale the method, a steady-state mathematical model was developed that predicts the process response to changes in input feed rate and counterflow or "moving bed" velocities. The vortex-stabilized apparatus used for the separation was modeled using four regions based on the different hydrodynamic flows in each section. Concentration profiles were then derived on the basis of the properties of the Piperoxan-sulfated beta-cyclodextrin system being studied. The effects of different regional flow rates on the concentration profiles were evaluated and used to predict the maximum processing rate and the hydrodynamic profiles required for a separation. Although the model was able to qualitatively predict the shapes of the concentration profiles and show where the theoretical limits of operation existed, it was not able to quantitatively match the data from actual enantiomer separations to better than 50% accuracy. This is believed to be due to the simplifying assumptions involved, namely, the neglect of electric field variations and the lack of a competitive binding isotherm in the analysis. Although the model cannot accurately predict concentrations from a separation, it provides a good theoretical framework for analyzing how the process responds to changes in counterflow rate, feed rate, and the properties of the molecules being separated.

Computer Simulation↗