Cornelius F. Ivory

Thermal model of capillary electrophoresis and a method for counteracting thermal band broadening

Thermal band broadening is known to be caused by the temperature dependence of ionic mobility. This dependence also strongly influences the temperature of the capillary by providing positive feedback between the temperature and power density. Previous thermal models of capillary electrophoresis have not fully considered this “autothermal effect”. We show that it always causes a capillary to run hotter than is predicted by a constant conductivity model; temperature excursions two times greater are typical. We propose that the thermally induced parabolic distortion of the migration velocity can be countered with an opposing Poiseuille (pressure-driven) flow. Dispersion calculations indicate that it may be possible to obtain plate numbers in excess of 106 m-1 even in very large bore (400 μm) capillaries.

Focusing proteins in an electric field gradient

Abstract Gradient techniques are a class of separation methods that combine the steps of isolation and concentration. This class of techniques uses one or more gradients of counteracting forces to create an ‘equilibrium point’ at which a protein can focus. Equilibrium gradient techniques can be adapted to a specific separation by choosing appropriate counteracting forces based on differences in the physical characteristics of proteins. The method introduced here, field gradient focusing, is an addition to this class of separation techniques which employs a gradient in the electric field to simultaneously separate and concentrate molecules.

Protein Focusing in a Conductivity Gradient

Conductivity gradient focusing (CGF) is one member of a family of gradient focusing techniques, characterized by two opposing forces which produce a dynamic equilibrium and which are able to simultaneously separate and concentrate proteins. In CGF, the two counteracting forces result from a constant convective flow of buffer opposed by an electric field gradient. This gradient in the electric field is formed by gradually decreasing buffer conductivity, i.e., when a slow‐moving, relatively high conductivity buffer is dialyzed against a low conductivity purge buffer. This paper presents the design of an analytical‐scale CGF device and the results of several experiments with colored proteins, both in free solution and with the use of a 45 μm size‐exclusion (SEC) packing to decrease dispersion. Experimental results with hemoglobin suggest that CGF may one day be capable of resolving proteins with small charge differences. A linear computer model of conductivity gradient focusing is derived, and some suggestions are made for further development of this new electrophoretic method.

A Brief Review of Alternative Electrofocusing Techniques

Isoelectric focusing (IEF) is an excellent tool at analytical scales but has some drawbacks at preparative and process scales. Alternative electrofocusing methods have been around for over a decade but have only recently reached the point where they can begin to compete head-to-head with IEF. This paper describes some of the advances made in this field since the mid-1980s and shows how they are related to IEF by a common mathematical expression. In addition, one new technique is described which allows real-time computer-control of the focusing gradient.

Field Gradient Focusing: A Novel Method for Protein Separation

Equilibrium gradient techniques constitute a class of separation methods that combine the steps of separation and concentration by using a gradient in one or more counteracting forces to create a stable equilibrium point at which a protein can focus. Different proteins focus at different equilibrium points, creating a steady‐state distribution of isolated proteins. Equilibrium gradient techniques can be adapted to a specific separation by choosing appropriate counteracting forces based on differences in the physical properties of the proteins involved. Zone electric field gradient focusing (FGF) is a new addition to this class of separation techniques with the unique property of using a gradient in the electric field to establish an equilibrium point instead of using a gradient in the velocity or pH. This paper presents two mathematical models which can be used to predict the steady‐state concentration profiles obtained by zone electric field gradient focusing. The first model applies only at very low protein concentrations where nonlinear effects can be ignored, e.g., less than 1 mg/mL, but it can be solved analytically and is useful in understanding the basic principles engendered in the method. The second model applies at all concentrations and allows for variations in the electric field strength where the protein focuses, but requires numerical solution. The design of an experimental device is also reported, as well as the results of two experiments: (1) the focusing of the protein hemoglobin from a dilute solution and (2) the separation of different oxidation states of the protein myoglobin.

Microfluidic isotachophoresis: a review.

Electromigration methods including CE and ITP are attractive for incorporation in microfluidic devices because they are relatively easily adaptable to miniaturization. After its popularity in the 1970s, ITP has made a comeback in microfluidic format (μ‐ITP, micro‐ITP) driven by the advantages of the steady‐state boundary, the self‐focusing effect, and the ability to aid in preconcentrating analytes in the sample while removing matrix components. In this review, we provide an overview of the developments in the area of μ‐ITP in a context of the historic developments with a focus on recent developments in experimental and computational ITP and discuss possible future trends. The chip‑ITP areas and topics discussed in this review and the corresponding sections include: PC simulations and modeling, analytical μ‐ITP, preconcentration ITP, transient ITP, peak mode ITP, gradient elution ITP, and free‐flow ITP, while the conclusions provide a critical summary and outlook. The review also contains experimental conditions for μ‐ITP applications to real‐world samples from over 50 original journal publications.

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.

10 000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the authors knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

Modeling biofilms with dual extracellular electron transfer mechanisms

Electrochemically active biofilms have a unique form of respiration in which they utilize solid external materials as terminal electron acceptors for their metabolism. Currently, two primary mechanisms have been identified for long-range extracellular electron transfer (EET): a diffusion- and a conduction-based mechanism. Evidence in the literature suggests that some biofilms, particularly Shewanella oneidensis, produce the requisite components for both mechanisms. In this study, a generic model is presented that incorporates the diffusion- and the conduction-based mechanisms and allows electrochemically active biofilms to utilize both simultaneously. The model was applied to S. oneidensis and Geobacter sulfurreducens biofilms using experimentally generated data found in the literature. Our simulation results show that (1) biofilms having both mechanisms available, especially if they can interact, may have a metabolic advantage over biofilms that can use only a single mechanism; (2) the thickness of G. sulfurreducens biofilms is likely not limited by conductivity; (3) accurate intrabiofilm diffusion coefficient values are critical for current generation predictions; and (4) the local biofilm potential and redox potential are two distinct parameters and cannot be assumed to have identical values. Finally, we determined that simulated cyclic and squarewave voltammetry based on our model are currently not capable of determining the specific percentages of extracellular electron transfer mechanisms in a biofilm. The developed model will be a critical tool for designing experiments to explain EET mechanisms.

The Prospects for Large-Scale Electrophoresis

Abstract Electrophoresis is capable of resolving biological materials on the basis of differences in their molecular weights, electrophoretic mobilities, isoelectric points, or various combinations of these properties. At laboratory scale, these include some of the most powerful techniques available for the gentle purification of biologically active molecules. For instance, isoelectric focusing (IEF, see Table 1) can resolve proteins whose isoelectric points, pIs, differ by as little as 0.01 pH unit. Likewise, SDS-PAGE will routinely isolate a discrete spectrum of proteins whose molecular weights differ by less than 2%. Although electrophoretic separations are sometimes carried out under hostile conditions, e.g., low salt concentrations, denaturing detergents, or extreme pHs, the basic technique is inherently mild and is universally applicable to the detection and purification of solutes ranging in size from several angstroms to several microns. Because of this, electrophoresis is widely used in the natur...