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. A method, comprising:
introducing, at a first time, a sample containing a plurality of analytes in a conductive medium into a capillary, ionically coupling a first end of the capillary to a first running buffer having a first pH;
ionically coupling a second end of the capillary to a second running buffer having a second pH, such that a pH gradient forms along the capillary;
separating, at a second time after the first time, at least a subset of the plurality analytes according to their isoelectric points by applying a voltage across the first running buffer and the second running buffer when the first end of the capillary is ionically coupled to the first running buffer and the second end of the capillary is ionically coupled to the second running buffer;
placing the second end of the capillary into a well including a chemical mobilizer at a third time after the first time to elute an analyte from the plurality of analytes from the capillary and into the well; and
applying a second voltage across the first running buffer and the chemical mobilizer at the third time and when the first end of the capillary is ionically coupled to the first running buffer and the second end of the capillary is disposed in the well, the second voltage and the chemical mobilizer collectively causing the analyte to be eluted.
2. The method of claim 1 , wherein a duration for which the second voltage is applied is based on a speed of mobilization associated with the analyte.
3. The method of claim 1 , further comprising:
increasing, at a fourth time after the third time, a pressure at the first end of the capillary such that the pressure induces hydrodynamic flow in the capillary, eluting the analyte.
4. The method of claim 3 , wherein an amount of the pressure applied at the first end of the capillary is based on a speed of mobilization associated with the analyte.
5. The method of claim 1 , wherein:
a metal tip is disposed at the second end of the capillary to provide electrical connectivity between the second end of the capillary and the first end of the capillary, such that the first voltage and the second voltage are applied via the metal tip.
6. The method of claim 1 , wherein a metal tip is disposed at the second end of the capillary, the method further comprising:
electrically coupling a first electrode to the first running buffer; and
electrically coupling a second electrode to the metal tip such that the first electrode, the second electrode, and a portion of the sample drawn into the capillary define a portion of an electrical circuit.
7. The method of claim 1 , wherein:
a porous membrane is disposed on the first end of the capillary, such that the porous membrane allows ion exchange between the first running buffer and the second running buffer when the second end of the capillary is disposed a running buffer reservoir containing the second running buffer while inhibiting hydrodynamic flow between the running buffer reservoir and the capillary.
8. The method of claim 1 , wherein the capillary has an inner diameter of 320-530 μm.
9. The method of claim 1 , wherein the capillary has an inner diameter of 200-500 μm.
10. The method of claim 1 , wherein the capillary has a length of 60-120 mm.
11. The method of claim 1 , wherein the capillary has a length of 20-30 cm.
12. The method of claim 1 , wherein:
a porous membrane configured to allow ion exchange between the first running buffer and the second running buffer and having a molecular weight cut-off from 10 kDa to 500 kDa is disposed on the first end of the capillary.
13. The method of claim 1 , wherein:
a porous membrane tubing configured to balance ion-exchange between the first running buffer and at least one of the second running buffer or the chemical mobilizer and having a molecular weight cut-off from 10 kDa to 500 kDa is disposed on the first end of the capillary.
14. The method of claim 1 , wherein:
a porous membrane tubing configured to balance ion-exchange between the first running buffer and at least one of the second running buffer or the chemical mobilizer and having a diameter of the porous membrane tubing that is matched with an inner and/or an outer diameter of the capillary is disposed on the first end of the capillary.
15. The method of claim 1 , wherein there is no porous membrane tubing disposed on the second end of the capillary.
16. The method of claim 1 , wherein:
the analyte is a first analyte from the plurality of analytes; and
separating at least the subset of the plurality of analytes causes the first analyte to be focused at a first distance from the second end of the capillary and a second analyte from the plurality of analytes to be focused at a second distance from the second end of the capillary, the second distance being greater than the first distance, the method further comprising:
determining a magnitude of the second voltage or a duration of application of the second voltage based on a measure of separation between the first distance and the second distance such that applying the second voltage over the duration causes the first analyte to be eluted into the well from the first distance, without the second analyte being eluted into the well.
17. The method of claim 16 , wherein the magnitude of the second voltage the chemical mobilizer the first analyte migrates towards the well at a rate 1 mm/min to 2 mm/min when the second voltage is applied.
18. An apparatus, comprising: The method of claim 1 , wherein:
the capillary is from at least one capillary tube that defines a capillary lumen, 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.
19. The method of claim 18 , wherein the first inner diameter is 2-3 times that of the second inner diameter.
20. The method of claim 18 , wherein the first inner diameter is between 300 and 500 μm.
21. The method of claim 18 , wherein the second inner diameter is between 100 and 200 μm.
22. The method of claim 18 , wherein the first length is 3-4 times that of the second length.
23. The method of claim 18 , wherein the at least one capillary tube that defines the capillary lumen is monolithically formed.
24. The method of claim 18 , 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.