Multi-point detection method for electrophoresis and chromatography in capillaries

• US 5,114,551 A
• Filed: 09/30/1991
• Issued: 05/19/1992
• Est. Priority Date: 09/30/1991
• Status: Expired due to Fees

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Srichaiyo, Tasanee, Hjerten, Stellan
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Koestner, Caroline
• Application Number:
  US07/769,073
• Time in Patent Office: 232 Days
• Field of Search: 204/299 R, 204/180.1, 204/182.8, 356/344, 436/161, 210/656, 210/198.2, 73/61.1 C, 73/23.4
• US Class Current: 204/452
• CPC Class Codes: G01N 2021/052 Tubular type; cavity type; multireflective
• CPC Class Codes: G01N 2030/6013 interfaces to detectors
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 30/6082 transparent to radiation
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J
• CPC Class Codes: G01N 30/8606 Integration

Abstract

Multiple electropherograms or chromatograms representing successive stages of an electrophoretic or chromatographic separation in a capillary are obtained by training a recorder on a succession of detection windows in the capillary, spaced apart along the capillary length. A single immovable detector is used, and the capillary is either held such that each detection window is placed in alignment with the detector in succession, or is looped such that the detection windows all lie in the path of the detector at the same time.

Claims

1. A method for obtaining a plurality of solute peak patterns representing successive stages of a continuous separation of solutes by a separation process selected from chromatographic and electrophoretic separations performed in a separation medium retained in a capillary, said method comprising detecting, with a single detector, variations in light absorptivity as a function of time at a plurality of locations spaced apart along the length of said capillary.

2. A method for determining values representative of the mobilities and relative amounts of solutes undergoing electrophoretic separation through a separation medium in a capillary, said method comprising:

(a) with a single detector, detecting variations in light absorptivity by said separation medium as a function of time at a plurality of detection points spaced apart along the length of said capillary;

(b) at each said detection point, identifying peaks in the variations in light absorptivity thus detected, and a sequence in which said peaks occur which is substantially the same at all said detection points;

(c) for each peak in said sequence, averaging among all said detection points the distance traveled by said peak to said detection point divided by the time taken by said peak to reach said detection point; and

(d) for each peak in said sequence, averaging among all said detection points the areas defined by peaks at the same position in said sequence.

3. A method for obtaining a plurality of solute peak patterns representing successive stages of a continuous separation of solutes by a separation process selected from the group consisting of chromatographic and electrophoretic separations performed in a separation medium retained in a capillary, as said solutes are migrating in a direction of migration in said capillary, said method comprising:

(a) aligning a first location in said capillary with a light absorptivity detector, and obtaining a first solute peak pattern by detecting variations in light absorptivity by said separation medium as a function of time at said first location;

(b) displacing said capillary relative to said detector to align therewith a second location further along the length of said capillary in said direction of migration, and obtaining a second solute peak pattern by detecting variations in light absorptivity as a function of time at said second location; and

(c) repeating step (b) at a sufficient number of locations to achieve said plurality of solute peak patterns.

4. A method in accordance with claim 3 in which said separation process is electrophoresis, and said solute peak patterns are electropherograms.

5. A method in accordance with claim 4 further comprising:

(d) for each of said electropherograms, identifying peaks therein and a sequence in which said peaks occur, said sequence being substantially the same in each of said electropherograms;

(e) for each peak in said sequence, averaging among all said electropherograms the distance traveled by said peak to the location at which said electropherogram is obtained divided by the time taken by said peak to reach said location; and

(f) for each peak in said sequence, averaging among all said electropherograms the areas defined by peaks at the same position in said sequence.

6. A method in accordance with claim 4 in which said capillary is a length of fused silica whose outer surface is coated with polyimide in a substantially continuous manner except for discontinuities at each of said locations aligned with said light absorptivity detector in steps (a) and (b).

7. A method in accordance with claim 6 in which the interior surface of said capillary is coated with a substance which suppresses adsorption and electroendosmosis and is transparent to ultraviolet light.

8. A method in accordance with claim 7 in which said substance is a member selected from the group consisting of linear polyacrylamide, dextran and methyl cellulose.

9. A method in accordance with claim 4 in which said capillary has an internal diameter of from about 5 microns to about 300 microns.

10. A method in accordance with claim 4 in which said capillary has an internal diameter of from about 20 microns to about 100 microns.

11. A method for obtaining a plurality of solute peak patterns representing successive stages of a continuous separation of solutes by a separation process selected from the group consisting of chromatographic and electrophoretic processes, said method comprising:

(a) subjecting said solutes to conditions promoting said separation process in a separation medium retained in a capillary, said capillary being looped at least once to place a plurality of locations along the length of said capillary defined as detection points in sufficient proximity to each other that all such detection points intercept a single detection beam of a light absorption detector, each pair of detection points separated by at least one loop of said capillary; and

(b) detecting variations in the absorption of light from said detection beam by said separation medium as a function of time, caused by the migration of said solutes through said separation medium past said detection points.

12. A method in accordance with claim 11 in which said separation process is chromatography, and said solute peak patterns are chromatograms.

13. A method in accordance with claim 11 in which said separation process is electrophoresis, and said solute peak patterns are electropherograms.

14. A method in accordance with claim 13 further comprising:

(c) identifying peaks in the variations thus detected, and grouping said peaks into electropherograms, one corresponding to each detection point;

(d) identifying a sequence in which said peaks occur in each of said electropherograms, said sequence being substantially the same in each of said electropherograms;

(e) for each peak in said sequence, averaging among all said electropherograms the distance traveled by said peak to the detection point at which said electropherogram is obtained divided by the time taken by said peak to reach said detection point; and

(f) for each peak in said sequence, averaging among all said electropherograms the areas defined by peaks at the same position in said sequence.

15. A method in accordance with claim 13 in which said capillary is a length of fused silica whose outer surface is coated with polyimide in a substantially continuous manner except for discontinuities at each of said detection points.

16. A method in accordance with claim 15 in which the interior surface of said capillary is coated with a substance which suppresses adsorption and electroendosmosis and is transparent to ultraviolet light.

17. A method in accordance with claim 16 in which said substance is a member selected from the group consisting of linear polyacrylamide, dextran and methyl cellulose.

18. A method in accordance with claim 11 in which said capillary has an internal diameter of from about 5 microns to about 300 microns.

19. A method in accordance with claim 11 in which said capillary

has an internal diameter of from about 20 microns to about 100 microns.

20. A method in accordance with claim 11 in which said detection beam is emanated from a light source and said variations in light absorption are detected by a light detector, and said detection points of said capillary, said light source and said light detector are all temperature controlled.