analyzing a solution; pumping fluid flow through channel ; applying electricity; measurement concentration
Inventors
Ross, David J. • Howell, Peter B. • Vreeland, Wyatt N.
Assignees
GOVERNMENT OF United States, COMMERCE NATIONAL INSTITUTE OF STANDARDS & TECHNOLOGY, Secretary of • National Institute of Standards and Technology NIST
Publication Number
US-7718046-B2
Publication Date
2010-05-18
Expiration Date
2024-06-10
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Abstract
A method and device are provided for affinity gradient focusing for directing at least one analyte in a solution containing a pseudostationary phase and located in a channel such as a capillary or a microchannel. The method includes establishing a steady-state spatial gradient in a retention factor of the pseudostationary phase for the at least one analyte. The analyte is caused to be moved within the channel whereby the concentration of the at least one analyte changes at one or more positions along the gradient. The pseudostationary phase is charged and the analyte is either neutral or charged or alternatively, the pseudostationary phase is neutral and the analyte is charged. The device may include a fluid channel, a pseudostationary phase having a retention factor gradient, an electrical current source and a pump system for establishing the bulk flow in the solution in the channel.
Core Innovation
The invention provides a novel method and device for affinity gradient focusing that directs one or more analytes in a solution containing a pseudostationary phase within a channel such as a capillary or microchannel. This method establishes a steady-state spatial gradient in the retention factor of the pseudostationary phase for the analyte, causing the analyte concentration to vary at one or more positions along the gradient. The method applies a combination of an electric field and bulk solution flow to move the analyte, exploiting the different affinities of analytes for the pseudostationary phase to achieve focusing and separation.
The problem addressed by the invention arises from limitations in prior art focusing techniques, which rely on electrophoretic mobility gradients and thus cannot separate neutral species or species with identical electrophoretic mobilities, such as chiral isomers. Traditional methods either lack a theoretical upper limit to concentration (focusing) or are limited in concentration factor (stacking). Micellar electrokinetic chromatography (EKC) provides separation based on affinity for a pseudostationary phase but does not achieve focusing where analytes accumulate at zero velocity points.
The invention uniquely combines focusing and EKC principles to produce a focusing method based on analyte affinity for a pseudostationary phase rather than solely on electrophoretic mobility. By establishing a steady-state retention factor gradient—via a temperature gradient or composition gradient—the analyte experiences spatially varying interactions resulting in a zero net velocity point where it accumulates. Charged or neutral pseudostationary phases and analytes of differing charge states can be used, enabling separation and concentration of neutral species and other analytes not separable by previous focusing methods.
Claims Coverage
The patent presents three independent claims that define various methods for directing analytes in solutions containing pseudostationary phases by establishing steady-state spatial gradients in retention factor and manipulating analyte movement using electric fields and bulk solution flow.
Method establishing a steady-state spatial gradient in pseudostationary phase retention factor using ionic pseudostationary phases
This method involves establishing a steady-state gradient in the retention factor of an ionic pseudostationary phase for an analyte (which may be neutral or charged), and moving the analyte by combining an electric field and bulk flow where the electric field drives the ionic pseudostationary phase electrophoretically from high to low retention factor regions and the bulk flow moves oppositely.
Method establishing steady-state retention factor gradient via composition gradient with neutral pseudostationary phases
This method creates a retention factor gradient by producing a gradient in solution composition and moves charged analytes using an electric field aligned with the gradient (from low to high retention factor region) with bulk solution flow in the opposite direction. The pseudostationary phase here is neutral.
Method applying electric field and bulk flow to ionic pseudostationary phases with steady-state spatial retention factor gradient
This method applies an electric field aligned with the retention factor gradient, directing ionic pseudostationary phases from high to low retention factor, and bulk solution flow opposite to that electrophoretic motion, with the pseudostationary phase being ionic and analytes either charged or neutral.
The independent claims collectively focus on methods to establish and maintain steady-state spatial gradients in retention factor within pseudostationary phases and to use combined electric fields and bulk flow to achieve focusing and separation of analytes based on their affinity for these phases. Variations encompass ionic and neutral pseudostationary phases, analyte charge states, and mechanisms for creating the retention factor gradient such as temperature or composition gradients.
Stated Advantages
Enables focusing and separation of neutral analytes that have zero electrophoretic mobility and cannot be separated by prior focusing methods.
Allows focusing based on properties other than electrophoretic mobility, broadening the range of analytes separable and concentratable.
Provides a focusing technique with no theoretical upper limit to analyte concentration, improving detection sensitivity.
Permits manipulation of the focused analyte position, enabling holding or moving the analyte for detection or additional analysis while maintaining focusing.
Uses steady-state gradients in retention factor, which unlike transient gradients in stacking or sweeping methods, can be maintained indefinitely under constant externally controlled parameters.
Integrates advantages of focusing with affinity-based separations like EKC, enabling concentration and separation of analytes that previous methods cannot achieve.
Potentially simple implementation using temperature gradients similar to temperature gradient focusing techniques.
Documented Applications
Preconcentration and separation of analytes, including neutral species and chiral isomers, in microfluidic chips and capillary systems.
Micro-total-analytical systems for movement, separation, reaction, and detection of various chemicals like proteins, DNA, and chemical compounds.
Chiral separations using chiral selectors combined with pseudostationary phases for spatially resolved focusing of enantiomers.
Two-dimensional separation schemes combining pseudostationary-phase-based focusing with other focusing techniques.
Use of temperature or composition gradients to induce retention factor gradients facilitating analyte focusing and separation.
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