Systems and methods for fractionation and collection of analytes in a sample
Embodiments include systems, apparatuses, and methods to efficiently separate analytes in a sample and elute fractions of the separated analytes. In some embodiments, a method includes introducing a sample in a capillary with a first end ionically coupled to a first running buffer and a second end ionically coupled to a second running buffer to form a pH gradient. The method includes applying a voltage between the first running buffer and the second running buffer, to separate a plurality of analytes in the sample. The method includes disposing the second end of the capillary in a collection well including a chemical mobilizer and applying a voltage to elute one or more analytes from the plurality of analytes in the sample, that have been separated, into the collection well. Embodiments include detection methods to monitor separation of analytes, mobilization of analytes, and/or elution of fractions containing analytes.
1. An apparatus, comprising:
a cartridge;
a capillary lumen at least partially disposed within the cartridge and defined by at least one capillary tube, the capillary lumen configured to contain an electrically conductive sample that includes a plurality of analytes, a first portion of the capillary lumen having a first inner diameter and a first length, and a second portion of the capillary lumen having a second inner diameter and a second length;
a running buffer reservoir disposed within the cartridge and configured to contain a first running buffer having a first pH, the capillary coupled to the running buffer reservoir such that a first end of the capillary lumen is configured to be ionically coupled to the first running buffer reservoir;
a sample plate defining a plurality of wells, the sample plate and the cartridge collectively configured such that a second end of the capillary lumen can be moved between each well from the plurality of wells;
a first well from the plurality of wells configured to contain a second running buffer having a second pH different from the first pH such that when the second end of the capillary lumen is disposed in the second running buffer, a pH gradient is established along the capillary lumen;
an electrical power source configured to apply a voltage across the running buffer reservoir and the second running buffer such that the plurality of analytes is focused according to their respective isoelectric points, generating a separated plurality of analytes;
a second well from the plurality of wells configured to contain a chemical mobilizer such that when the second end of the capillary lumen is disposed in the second well, at least one analyte from the separated plurality of analytes is mobilized into and collected in the second well.
2. The apparatus of claim 1 , wherein the first inner diameter is 2-3 times that of the second inner diameter.
3. The apparatus of claim 1 , wherein the first inner diameter is between 300 and 500 μm.
4. The apparatus of claim 1 , wherein the second inner diameter is between 100 and 200 μm.
5. The apparatus of claim 1 , wherein the first length is 3-4 times that of the second length.
6. The apparatus of claim 1 , wherein the at least one capillary tube is monolithically formed.
7. The apparatus of claim 1 , wherein the at least one capillary tube includes a first capillary tube associated with the first portion of the capillary lumen joined to a second capillary tube associated with a second portion of the capillary lumen.
8. The apparatus of claim 1 , further comprising:
a metal tip disposed over the second end of the capillary lumen.
9. The apparatus of claim 8 , further comprising:
a first electrode electrically coupled the running buffer reservoir and configured to be electrically coupled to the first running buffer; and
a second electrode electrically coupled to the metal tip such that the first electrode, the second electrode, and the electrically conductive sample define a portion of an electrical circuit.
10. The apparatus of claim 1 , further comprising:
a porous membrane disposed on the first end of the capillary lumen, the porous membrane being configured to allow ion exchange between the first running buffer and the second running buffer when the second end of the capillary lumen is disposed in the first well, the porous membrane further configured to inhibit hydrodynamic flow between the running buffer reservoir and the capillary lumen.
11. The apparatus of claim 10 , wherein the porous membrane has a molecular weight cut-off from 10 kDa to 500 kDa.
12. The apparatus of claim 1 , wherein there is no porous membrane tubing disposed on the second end of the capillary lumen.
13. The apparatus of claim 1 , wherein the voltage is a first voltage; and the electrical power source is further configured to apply a second voltage across the running buffer reservoir and the chemical mobilizer when the second end of the capillary lumen is disposed in the second well such that the analyte migrates into the second well from capillary lumen.
14. The apparatus of claim 13 , wherein the analyte migrates toward the second well at a rate of 1 mm/min to 2 mm/min when the second voltage is applied and when the second end of the capillary lumen is disposed in the second well.
15. The apparatus of claim 1 , wherein the voltage is a first voltage; and wherein a first analyte of the plurality of analytes is focused at a first distance from the second end of the capillary lumen and a second analyte of the plurality of analytes is focused at a second distance from the second end of the capillary lumen when the first voltage is applied; wherein the second distance is greater than the first distance; and
wherein the electrical power source is further configured to apply a second voltage across the running buffer reservoir and the chemical mobilizer when the second end of the capillary lumen is disposed in the second well such that the first portion migrates into the second well from the first distance; and
wherein a magnitude of the second voltage is determined based on a measure of separation between the first distance and the second distance.
16. The apparatus of claim 1 , wherein the first inner diameter is larger than the second inner diameter.
17. The apparatus of claim 16 , wherein the first length is larger than the second length.
18. The apparatus of claim 1 , further comprising a transition portion of the capillary lumen located between the first portion of the capillary lumen and the second portion of the capillary lumen.