Systems and methods for two-dimensional chromatography

Patent metadata

Field	Value
Publication number	US-10788467-B2
Application number	US-201816125427-A
Country	US
Kind code	B2
Filing date	Sep 7, 2018
Priority date	Oct 27, 2014
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

Abstract

Provided are two-dimensional chromatography systems and methods for separating and/or analyzing complex mixtures of organic compounds. In particularly, a two-dimensional reversed-phase liquid chromatography (RPLC)—supercritical fluid chromatography (SFC) system is described including a trapping column at the interface which collects the analytes eluted from the first dimension chromatography while letting the RPLC mobile phase pass through. The peaks of interest from the RPLC dimension column are effectively focused as sharp concentration pulses on the trapping column, which is subsequently injected onto the second dimension SFC column. The system can be used for simultaneous achiral and chiral analysis of pharmaceutical compounds. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess).

First claim

What is claimed is:

1. A method for separating a sample, the method comprising the steps of:
(i) capturing a first mobile phase comprising at least a portion of the sample on a trapping column,
wherein the first mobile phase is a reversed-phase liquid chromatography (RPLC) mobile phase,
wherein said portion is obtained by separating the sample by a RPLC, and
wherein said trapping column comprises a stationary phase; and

(ii) subjecting the RPLC mobile phase comprising the portion of the sample captured on the trapping column to further separation by a supercritical fluid chromatography (SFC).

2. The method of claim 1 , further comprising separating the sample with the RPLC, wherein the RPLC comprises:
(i) introducing the sample into a first mobile phase;
(ii) driving the first mobile phase containing the sample through a RPLC column; and
(iii) separating the sample on the RPLC column.

3. The method of claim 2 , further comprising detecting the presence of a component of the sample in the first mobile phase after passing through the RPLC column.

4. The method of claim 2 , wherein the RPLC column comprises a reversed-phase stationary phase.

5. The method of claim 1 , further comprising eluting the portion of the sample captured on the trapping column off the trapping column.

6. The method of claim 1 , further comprising detecting a component of the sample after further separation by the SFC.

7. The method of claim 1 , further comprising positioning the trapping column in a flow path of the first mobile phase downstream of a RPLC column for capturing the portion of the sample separated by the RPLC column.

8. The method of claim 7 , further comprising switching the trapping column carrying the captured portion to a flow path of a second mobile phase for eluting the captured portion off the trapping column.

9. The method of claim 8 , wherein the step of positioning the trapping column in the flow path of the first mobile phase or the step of switching the trapping column to the flow path of the second mobile phase is performed in a fluidic routing unit interfacing a fluidic path of the RPLC and a fluidic path of the SFC.

10. The method of claim 1 , further comprising re-capturing at least some of the portion of the sample eluted off the trapping column on a focus column prior to further separation by the SFC.

11. The method of claim 1 , wherein the first mobile phase flows through the trapping column in a first direction, and the portion of the sample captured on the trapping column is eluted off the trapping column by flowing a second mobile phase through the trapping column in a direction opposite to the first direction.

12. The method of claim 1 , wherein the first mobile phase flows through the trapping column in a first direction, and the portion of the sample captured on the trapping column is eluted off the trapping column by flowing a second mobile phase through the trapping column in a direction same as the first direction.

13. The method of claim 1 , comprising the steps of:
(i) introducing a sample into the first mobile phase;
(ii) driving the first mobile phase containing the sample through a RPLC column;
(iii) separating the sample on the RPLC column;
(iv) detecting the presence of a component of the sample in the first mobile phase after passing through the RPLC column;
(v) capturing on the trapping column at least a first portion of the sample separated on the RPLC column;
(vi) eluting the first portion of the sample captured on the trapping column off the trapping column;
(vii) subjecting the first portion of the sample captured on the trapping column to further separation by the SFC; and
(viii) detecting a component of the sample after further separation by the SFC.

14. The method of claim 13 , further comprising the steps of:
(ix) capturing on a second trapping column at least a second portion of the sample separated on the RPLC column, said second trapping column comprising a stationary phase;
(x) eluting the second portion of the sample captured on the second trapping column off the second trapping column;
(xi) subjecting the second portion of the sample captured on the second trapping column to further separation by the SFC.

15. The method of claim 1 , wherein the stationary phase in the trapping column comprises a reversed-phase material.

16. The method of claim 1 , wherein further separation by SFC is performed on a SFC column comprising a normal phase stationary phase.

17. The method of claim 1 , wherein further separation by the SFC is performed on a SFC system comprising an array of SFC columns.

18. The method of claim 17 , wherein each of the SFC columns independently comprises a normal phase stationary phase.

19. The method of claim 18 , further comprising routing the portion of the sample captured on the trapping column to a SFC column for further separation, said SFC column comprises a stationary phase adapted for separating components in the sample.

20. The method of claim 1 , wherein the volume of the RPLC mobile phase comprising the portion of the sample subjected to further separation by the SFC is about 10 μL to about 100 μL.

21. The method of claim 1 , wherein the volume of the RPLC mobile phase comprising the portion of the sample subjected to further separation by the SFC is about 30 μL or less.

22. A method for analyzing a sample using a chromatography system, the method comprising:
separating the sample into a first set of fractions by a reversed-phase liquid chromatography (RPLC) on a first separation unit; and
further separating one or more of the fractions by a supercritical fluid chromatography (SFC) on a second separation unit,
wherein the chromatography system comprises a first fluidic routing unit comprising a plurality of sample loops, said first fluidic routing unit is connected to the first separation unit and the second separation unit,
wherein at least one of the plurality of sample loops comprises a trapping column, said trapping column comprising a stationary phase, and
wherein the chromatography system is configured to analyze the sample using the chromatography system by for first separating the sample in the first separation unit and subsequently introducing at least a portion of the sample eluted from the RPLC column of the first separation unit to the second separation unit.

23. The method of claim 22 , wherein separation by the RPLC on the first separation unit is based in part on a first characteristic of the sample and separation by the SFC on the second separation unit is based in part on a second characteristic of the sample, said second characteristic of the sample is different from the first characteristic of the sample.

24. The method of claim 22 , wherein the sample comprises a mixture of stereoisomeric components.

25. The method of claim 24 , wherein the stereoisomeric components are separated into one or more fractions by the RPLC on the first separation unit, each of said fraction comprising an enantiomeric pair.

26. The method of claim 25 , wherein separation by the RPLC on the first separation unit is based in part on the hydrophobicity of the complex sample.

27. The method of claim 25 , wherein the enantiomeric pair is further separated into individual enantiomers by the SFC on the second separation unit.

28. The method of claim 27 , wherein separation by the

Assignees

• Genentech Inc

Inventors

• Venkatramani Cadapakam
• Al-Sayah Mohammad

Classifications

• G01N30/46 Primary

  using more than one column {(G01N30/44 takes precedence)} · CPC title

• G01N2030/207

  with metering cavity, e.g. sample loop · CPC title

• G01N2030/202

  rotary valves · CPC title

• G01N2030/201

  multiport valves, i.e. having more than two ports · CPC title

• B01D15/14 Primary

  relating to the introduction of the feed to the apparatus · CPC title