System for sample detection with compensation for difference in sensitivity to detection of components moving at different velocities

• US 5,503,994 A
• Filed: 10/08/1993
• Issued: 04/02/1996
• Est. Priority Date: 10/08/1993
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Shear, Jason B., Fishman, Harvey A., Dadoo, Rajeev, Shafer, Neil, Zare, Richard N.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/134,290
• Time in Patent Office: 907 Days
• Field of Search: 435/4, 435/6, 436/53, 436/164, 436/172, 436/171, 436/94, 436/86, 436/89, 436/90, 422/50, 422/52, 422/68.1, 422/70, 422/82.5, 422/82.08, 422/82.09, 356/344, 204/182.8
• US Class Current: 436/90
• CPC Class Codes: G01N 2030/324 speed, flow rate
• CPC Class Codes: G01N 2030/621 signal-to-noise ratio
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 30/0005 Field flow fractionation
• CPC Class Codes: G01N 30/62 Detectors specially adapted therefor
• 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 35/085 Flow Injection Analysis
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream

Abstract

In many separation techniques, such as field flow fractionation, liquid chromatography and electro-phoresis, chemical species form bands that migrate at different velocities. If the data-digitization rate and excitation intensity are both set to be optimal for the fastest migrating band, to compensate for different band velocities, both the data-digitization rate and the excitation intensity are decreased as a function of time by a factor equal to the migration time of the fastest migrating band to the separation time.

Claims

1. A method for separation and detection of components of a sample, said method comprising the steps of:

causing components of the sample to migrate at different velocities and separate in a separation path;

supplying radiation to the components to cause the components to exhibit a detectable response, wherein fractions of said components are altered by the radiation so as to become undetectable;

detecting the response of the components when they are migrating at different velocities at a data rate;

wherein the intensity of the radiation supplied in the supplying step or the data rate of the detecting step is altered as a function of different lengths of migration time of the components to reduce differences in signal-to-noise ratio of components migrating at different velocities.

2. The method of claim 1, said supplying step further comprising the step of setting the intensity of radiation supplied to the sample to a predetermined value before radiation is supplied to the sample, wherein said intensity of radiation supplied is decreased from said predetermined value as a function of length of migration time.

3. The method of claim 2, wherein said detecting step includes setting the data rate of the response from the components of the sample to a second predetermined value for detecting a particular component of the sample, wherein said data rate is decreased from said predetermined value as a function of length of migration time.

4. The method of claim 2, wherein said setting step sets the intensity of radiation supplied to the sample to an optimum value for detecting a particular component of the sample, wherein the intensity of radiation is reduced to a value optimum for another component of the sample.

5. The method of claim 4, wherein said setting step sets the intensity of radiation supplied to the sample to an optimum value for detecting the fastest component of the sample, and wherein the intensity of radiation supplied is reduced from said optimum value as a function of length of migration time of said another component.

6. The method of claim 3, wherein said setting step sets the intensity of radiation supplied to the sample to substantially (5/4)(1/σ

*φ

), where σ

is the absorption cross-section of the particular component, and φ

is the radiation-alteration quantum efficiency of the particular component.

7. The method of claim 1, wherein said detecting step includes setting the data rate of the response from the components of the sample to an optimum value for detecting a particular component of the sample.

8. The method of claim 7, further comprising reducing the data rate during the migration and separation of the sample components and during the detecting step.

9. The method of claim 7, further comprising reducing the data rate after the detecting step.

10. The method of claim 1, said supplying step including providing a source of radiation of substantially constant intensity and attenuating the radiation from said source as a function of length of migration time of the components to reduce differences in signal-to-noise ratio of components migrating at different velocities.

11. The method of claim 1, further comprising altering the data rate of response detected after the detecting step.

12. The method of claim 1, wherein said components exhibit a response according to one of the following mechanisms in response to the radiation supplied:

fluorescence or absorption, and said detecting step detects the fluorescence or absorption response of the components.

13. The method of claim 1, further comprising labeling the sample with a fluorophore prior to the causing step.

14. The method of claim 1, said causing step including applying a separation gradient to the sample in a separation medium.

15. The method of claim 14, wherein said applying step applies an electrical potential gradient across the separation medium.

16. The method of claim 1, wherein said supplying step applies electromagnetic radiation to the sample.

17. A method for detection of components of a sample migrating at different velocities along a path, said method comprising the steps of:

supplying radiation to the components to cause the components to exhibit a detectable response, wherein fractions of said components are altered by the radiation so as to become undetectable;

detecting the response of the components when they are migrating at different velocities at a data rate;

wherein the intensity of the radiation supplied in the supplying step or the data rate of the detecting step is altered as a function of different lengths of migration time of the components to reduce differences in signal-to-noise ratio of components migrating at different velocities.

18. The method of claim 17, said supplying step further comprising setting the intensity of radiation supplied to the sample to a predetermined value before radiation is supplied to the sample, wherein said intensity of radiation supplied is decreased from said predetermined value as a function of length of migration time.

19. The method of claim 18, wherein said setting step sets the intensity of radiation supplied to the sample to an optimum value for detecting a particular component of the sample.

20. The method of claim 19, wherein said setting step sets the intensity of radiation supplied to the sample to an optimum value for detecting the fastest component of the sample, and wherein the intensity of radiation supplied is reduced from said predetermined value as a function of length of migration time.

21. The method of claim 19, wherein said setting step sets the intensity of radiation supplied to the sample to substantially (5/4)(1/σ

*φ

), where σ

is the absorption cross-section of the particular component, and φ

is the radiation-alteration quantum efficiency of the particular component.

22. The method of claim 17, wherein said detecting step includes setting the data rate of the response from the components of the sample to an optimum value for detecting a particular component of the sample.

23. The method of claim 22, wherein said setting step sets the data rate of the response from the components of the sample to an optimum value for detecting the fastest component of the sample, and wherein the data rate of detection of components of the sample is reduced from said predetermined value as a function of length of migration time.

24. The method of claim 23, wherein the data rate of detection of components of the sample is reduced during the detecting step.

25. The method of claim 23, wherein the data rate of detection of components of the sample is reduced after the detecting step.

26. The method of claim 17, said supplying step including providing a source of radiation of substantially constant intensity and attenuating the radiation from said source as a function of length of migration time of the components to reduce differences in signal-to-noise ratio of components migrating at different velocities.

27. The method of claim 17, further comprising altering the data rate of response detected after the detecting step.

28. The method of claim 17, wherein said components exhibit a response according to one of the following mechanisms in response to the radiation supplied:

fluorescence or absorption, and said detecting step detects the fluorescence or absorption response of the components.

29. The method of claim 17, further comprising labeling the sample with a fluorophore prior to the supplying step.

30. The method of claim 17, wherein said supplying step applies electromagnetic radiation to the sample.

31. A method for detection of components of a sample migrating at different velocities along a path, said method comprising the steps of:

supplying radiation to the components in a detection zone of the path to cause the components to exhibit a detectable response, wherein fractions of said components are altered by the radiation so as to become undetectable;

detecting the response of the components when they are migrating in the zone at different velocities at a data rate;

wherein the intensity of the radiation supplied in the supplying step or the data rate of the detecting step is altered as a function of different lengths of migration time of the components in the zone to reduce differences in signal-to-noise ratio of components migrating at different velocities.