Gradient elution electrophoresis
Inventors
Ross, David J. • Shackman, Jonathan G.
Assignees
GOVERNMENT OF UNITED STATED OF AMERICA COMMERCE NATIONAL INSTITTUTE OF STANDARDS AND TECHNOLOGY, Secretary of • National Institute of Standards and Technology NIST
Publication Number
US-8080144-B2
Publication Date
2011-12-20
Expiration Date
2027-10-03
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Abstract
A method for performing electrophoretic separation of ionic compounds which involves varying a bulk fluid flow though a separation path into which ionic species are continuously introduced and separated. The method can also include the introduction of a leading electrolyte into the separation path to form an ionic interface with the sample and an optional terminating electrolyte to enrich ionic species for higher detection resolution.
Core Innovation
The invention provides a method for electrophoretic separation of compounds that involves a separation path with a sample continuously in fluid contact at the inlet end and a separation buffer at the outlet end. An electric field is applied along the separation path to drive the electrokinetic motion of the compounds, while the bulk fluid flow through the separation path is varied over time concurrently with the electric field. This variation in bulk flow controls whether and when each compound moves into and through the separation path, enabling compounds with distinct electrophoretic mobilities to be introduced at different times.
This method overcomes limitations of prior art capillary isotachophoresis (cITP) and capillary electrophoresis (CE) methods that require discrete injections and long separation channels, resulting in larger device footprints and lower resolution in microfluidic chip formats. By continuously introducing sample and varying bulk flow, the invention enables high resolution electrophoretic separations in short columns, suitable for microfluidic applications, without the need for injection mechanisms or long serpentine channels.
An embodiment includes introducing leading electrolytes to establish ionic interfaces with the sample and optionally including terminating or spacing electrolytes to enhance concentration and resolution. The bulk fluid flow can be driven or varied by pressure gradients and/or electroosmotic flow. The method also applies electrophoretic separation under isotachophoretic conditions with continuous sample introduction and variable bulk flow for enhanced enrichment and separation, termed gradient elution moving boundary electrophoresis (GEMBE) and gradient elution isotachophoresis (GEITP).
Claims Coverage
The patent contains two independent method claims and covers the inventive features related to the electrophoretic separation with continuous sample contact combined with varying bulk fluid flow.
Electrophoretic separation with continuous sample contact and variable bulk flow
The method provides a separation path with continuous fluid contact of the sample at the inlet end and separation buffer at the outlet end, applies an electric field along the path to drive electrokinetic motion, and varies bulk fluid flow through the separation path over time concurrent with the electric field to control the timing of compound entry and migration, ensuring distinct compounds enter at different times.
Bulk fluid flow variation in a substantially continuous manner
The method includes varying the bulk fluid flow with respect to time in a substantially continuous manner, including linearly with constant acceleration or non-linearly with non-constant acceleration, as the sole means to affect whether and when compounds enter the separation path.
Use of leading electrolyte to concentrate compounds
The method uses a leading electrolyte in the separation buffer to concentrate compounds, optionally supplemented with terminating electrolytes and spacing electrolytes in the sample to provide isotachophoretic termination or spacers for enhanced concentration and resolution.
The independent claims cover the method of electrophoretic separation using continuous sample introduction combined with controlled and time-varying bulk fluid flow under an applied electric field, with features including continuous variation of bulk flow, use of leading electrolytes for concentration, and the option of isotachophoretic termination and spacing electrolytes to improve analyte resolution and enrichment.
Stated Advantages
Requires much shorter separation lengths and allows a much smaller total footprint on a microfluidic chip compared to prior art methods.
Eliminates the need for injection mechanisms, reducing costs and fabrication complexity for microfluidic devices.
Enables higher resolution separations due to the ability to vary bulk flow acceleration precisely, addressing electrode and electroosmotic flow variability issues.
Allows integration of more analytical elements within a fixed chip area due to reduced electrophoretic separation area requirements.
Facilitates high analyte concentration enhancement and high resolution separations in short lengths without buffer or polarity switching, improving reliability and automation.
Insensitivity to salts or ionic impurities in the sample matrix, which can improve resolution in isotachophoretic embodiments.
Documented Applications
Separation and enrichment of ionic compounds including small molecules, amino acids, proteins, nucleic acids, carbohydrates, peptides, pesticides, pharmaceuticals, cells, viruses, bacteria, nanoparticles, and particles in microfluidic and capillary systems.
Use in microfluidic chips for trace analysis with small total footprint areas.
Employing continuous sample introduction and variable hydrodynamic flow for electrophoretic separations in capillaries and microfluidic channels.
Gradient elution moving boundary electrophoresis (GEMBE) for high resolution separations with short columns and continuous sample input.
Gradient elution isotachophoresis (GEITP) for enrichment and separation of analytes at ionic interfaces formed by leading and terminating electrolytes.
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