Use of zwitterions to mobilize isoelectrically focused ampholyte zones

• US 5,110,434 A
• Filed: 12/20/1990
• Issued: 05/05/1992
• Est. Priority Date: 12/20/1990
• Status: Expired due to Term

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Rodriguez, Roberto, Wehr, C. Timothy, Zhu, Minge-De
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Koestner, Caroline
• Application Number:
  US07/633,646
• Time in Patent Office: 502 Days
• Field of Search: 204/182.9, 204/183.2, 204/183.3
• US Class Current: 204/451
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44795 Isoelectric focusing

Abstract

Ampholytic solute zones which have been stabilized by isoelectrical focusing in a separation medium such as a capillary are mobilized by the introduction of a relatively large quantity of zwitterion to one of the electrode chambers. When a voltage is then applied between the electrode chambers as in the focusing stage, the zwitterion migrates into the separation medium in accordance with the isoelectric point of the zwitterion. The voltage is maintained to caused continuous migration of the zwitterion into the capillary, thereby forming an expanding zone which displaces the focused zones formed in the focusing stage and causes them to travel axially through the capillary toward a detection point or out of the capillary for purposes of recovery.

Claims

1. A method for the mobilization of zones of ampholyte substances isoelectrically focused in an ampholytic separation medium between an anolyte at a first pH and a catholyte at a second pH higher than said first pH, said method comprising:

(a) introducing into either said anolyte or said catholyte an ampholyte having an isoelectric point between said first and second pH'"'"'s and defined as a mobilization ampholyte; and

(b) applying a voltage between said anolyte and said catholyte to cause migration of said mobilization ampholyte into said ampholytic separation medium, and maintaining said voltage to form a continuously expanding zone of said mobilization ampholyte in said ampholytic separation medium, thereby displacing each of said isoelectrically focused zones by said expanding zone past a locus of detection.

2. A method in accordance with claim 1 in which the isoelectric points of said ampholyte substances of said isoelectrically focused zones define a range of values, and the isoelectric point of said mobilization ampholyte is outside of said range of values.

3. A method in accordance with claim 2 in which the isoelectric point of said mobilization ampholyte is below said range.

4. A method in accordance with claim 2 in which the isoelectric point of said mobilization ampholyte is about 4.0 or less, and step (a) comprises introducing said mobilization ampholyte into said catholyte.

5. A method in accordance with claim 2 in which the pH of said anolyte is about 2 or below, the pH of said catholyte is about 12 or above, the isoelectric point of said mobilization ampholyte is from about 2.1 to about 4.0, and step (a) comprises introducing said mobilization ampholyte into said catholyte.

6. A method in accordance with claim 2 in which the isoelectric point of said mobilization ampholyte is above said range.

7. A method in accordance with claim 2 in which the isoelectric point of said mobilization ampholyte is about 9.0 or higher, and step (a) comprises introducing said mobilization ampholyte into said anolyte.

8. A method in accordance with claim 2 in which the pH of said anolyte is about 3 or below, the pH of said catholyte is about 12 or above, the isoelectric point of said mobilization ampholyte is from about 9.0 to about 11.9, and step (a) comprises introducing said mobilization ampholyte into said anolyte.

9. A method in accordance with claim 1 in which said mobilization ampholyte is an amino acid.

10. A method in accordance with claim 1 in which said ampholytic separation medium is a liquid solution in a capillary having an internal diameter of 500 microns or less.

11. A method in accordance with claim 1 in which said ampholytic separation medium is a liquid solution in a capillary having an internal diameter of 100 microns or less.