Temperature-assisted on-column focusing

Patent metadata

Field	Value
Publication number	US-12576344-B2
Application number	US-201916446287-A
Country	US
Kind code	B2
Filing date	Jun 19, 2019
Priority date	Jan 16, 2015
Publication date	Mar 17, 2026
Grant date	Mar 17, 2026

Abstract

A method comprising:
introducing a sample volume into an inlet end of a liquid chromatography column, wherein the liquid chromatography column includes a focusing segment proximal to the inlet end of the liquid chromatography column and a separation segment proximal to an elute outlet of the liquid chromatography column; maintaining only the focusing segment at a first temperature as the sample is introduced into the focusing segment; and subsequently heating the focusing segment to a second temperature that is higher than the first temperature after the entire sample volume has been introduced into the focusing segment.

First claim

What is claimed is:

1 . A method comprising:
introducing a sample having a predetermined volume into an inlet end of a liquid chromatography column, wherein the liquid chromatography column includes a first focusing segment proximal to the inlet end of the liquid chromatography column, a separation segment proximal to an elute outlet of the liquid chromatography column, and at least one second focusing segment located between the first focusing segment and the separation segment; maintaining the first focusing segment and the at least one second focusing segment at a first temperature as the sample is introduced into the first focusing segment; subsequently heating the first focusing segment to a second temperature that is higher than the first temperature while maintaining the at least one second focusing segment at the first temperature, wherein the subsequent heating of the first focusing segment is initiated after all of the predetermined sample volume has passed into the first focusing segment; subsequently heating the at least one second focusing segment to a third temperature that is higher than the first temperature; and heating the separation segment to the second temperature or the third temperature.

2 . The method of claim 1 , wherein the second temperature is at least 20° C. greater than the first temperature.

3 . The method of claim 1 , wherein maintaining the first focusing segment at a first temperature comprises cooling the first focusing segment for less than 30 seconds.

4 . The method of claim 1 , wherein the separation segment is not cooled.

5 . The method of claim 1 , wherein the liquid chromatography column is a capillary liquid chromatography column.

6 . The method of claim 1 , wherein separation of the sample volume is completed in less than three minutes.

7 . The method of claim 1 , wherein the first focusing segment and the second focusing segment each contain particles having an average particle diameter that is greater than the average particle diameter of particles contained in the separation segment.

8 . The method of claim 1 , further comprising introducing the sample volume into an inlet segment that is adjacent to the inlet end of the liquid chromatography column prior to introducing the sample volume into the first focusing segment.

9 . The method of claim 8 , wherein the inlet segment contains a sample injection element and noninteracting, nonporous silica spheres.

10 . The method of claim 1 , wherein the first focusing segment has a length that is 1 to 50 percent of the total length of the sum of the first focusing segment length, the second focusing segment length, and the separation segment length.

11 . The method of claim 1 , wherein the sample is associated with a mobile phase and the method further comprises applying a constant pressure to the mobile phase throughout a retention time period of the sample volume in the first focusing segment, second focusing segment, and the separation segment.

12 . The method of claim 1 , further comprising diluting the sample with a weak solvent.

13 . The method of claim 1 , wherein the first temperature is 30° C. to −20° C.

14 . The method of claim 2 , wherein maintaining the first focusing segment at a first temperature comprises cooling the first focusing segment for less than 30 seconds.

15 . The method of claim 2 , wherein the first temperature is 30° C. to −20° C.

16 . The method of claim 11 , wherein separation of the sample volume is completed in less than three minutes.

17 . The method of claim 1 , wherein the second focusing segment has a length that is 1 to 50 percent of the total length of the sum of the first focusing segment length, the second focusing segment length, and the separation segment length.

18 . The method of claim 1 , wherein the second temperature and the third temperature are the same.

19 . The method of claim 1 wherein the second temperature and the third temperature are different.

20 . The method of claim 1 , wherein heating the first focusing segment to a second temperature comprises heating the first focusing segment for less than 1000 seconds.

21 . The method of claim 1 , wherein heating the first focusing segment to a second temperature comprises heating the first focusing segment for less than 60 seconds.

22 . The method of claim 2 , wherein heating the first focusing segment to a second temperature comprises heating the first focusing segment for less than 1000 seconds.

23 . The method of claim 2 , wherein heating the first focusing segment to a second temperature comprises heating the first focusing segment for less than 60 seconds.

24 . The method of claim 1 , wherein the separation segment is heated to the second temperature.

25 . The method of claim 1 , wherein the separation segment is heated to the third temperature.

26 . The method of claim 1 , wherein the method occurs in a single column.

27 . The method of claim 1 , wherein the second temperature is at least 30° C. greater than the first temperature.

28 . The method of claim 1 , wherein the second temperature is at least 50° C. greater than the first temperature.

Assignees

• Univ Of Pittsburgh—Of The Commonwealth System Of Higher Education

Inventors

• Weber Stephen
• Groskreutz Stephen Robert
• Horner Anthony Robert
• Wilson Rachael-Anh Elizabeth

Classifications

• G01N2030/167

  on-column injection · CPC title

• G01N30/6039

  in series · CPC title

• G01N2030/3053

  using resistive heating · CPC title

• B01D15/325

  Reversed phase · CPC title

• B01D15/206

  Packing or coating · CPC title