Signal processing for chemical analysis of samples

• US 5,498,875 A
• Filed: 08/17/1994
• Issued: 03/12/1996
• Est. Priority Date: 08/17/1994
• Status: Expired due to Term

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Silzel, John W., Obremski, Robert J.
• Primary Examiner(s): FIELDS, CAROLYN
• Application Number:
  US08/292,798
• Time in Patent Office: 573 Days
• Field of Search: 250/458.1, 250/459.1, 356/301, 356/73, 356/317, 356/318
• US Class Current: 250/458.1
• CPC Class Codes: G01J 3/4338 Frequency modulated spectrometry
• CPC Class Codes: G01N 21/314 with comparison of measurements at specific and non-specific wavelengths dual wavelength spectrometry G01J3/427
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2201/1293 resolving multicomponent spectra

Abstract

A method and apparatus determines the analyte content of a sample by generating first and second input signals and directing the input signals to the sample. The input signals differ in wavelength by at least 3 nanometers. Due to the interaction between the input signals and the sample, first and second output signals are generated. Each output signal comprises a resonant signal whose peak wavelength is substantially independent of the wavelength of the respective input signal, and a non-resonant output signal whose peak wavelength is dependent upon the wavelength of respective input signal. A detector is used to detect the two output signals, and by distinguishing the resonant output signals from the non-resonant output signals, data about the analyte content of the sample is determined. Principal components regression analysis or multivariate quantitative analysis can be applied to the output signals, for the purpose of distinguishing between the resonant and non-resonant signals. The method and apparatus can also distinguish resonant output signals from each other, and non-resonant output signals from each other.

Claims

1. Apparatus for determining the analyte content of a sample, the apparatus comprising:

(a) a signal generator for generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) a means for directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal and (ii) a first non-resonant output signal whose peak wavelength is dependent upon the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal;

(c) a detector for detecting the first and second output signals; and

(d) means for distinguishing the resonant output signals from the non-resonant output signals to obtain data about the analyte content of the sample.

2. An apparatus as claimed in claim 1 wherein the signal generator comprises:

(i) two separate sources, each one for generating a source signal;

(ii) a modulator for selectively directing the source signals to the sample as the input signals; and

(iii) means for directing the source signals to the modulator.

3. Apparatus as claimed in claim 2 wherein the sources are laser diodes operative at different wavelengths.

4. An apparatus as claimed in claim 2 wherein the modulator comprises a wavelength modulating device and the means for directing the first and second input signals comprises a fiberoptic waveguide having a common waveguide and two limbs, each limb being directed to one of the sources, with the common waveguide being directed to the wavelength modulating device.

5. Apparatus as claimed in claim 4 wherein the wavelength modulating device is a liquid crystal filter.

6. Apparatus as claimed in claim 1 including processing means between the sample and the detector, the processing means including at least one of a filter, dispersing element and monochromator.

7. Apparatus as claimed in claim 6 wherein the processing means includes the filter, the dispersing element and the monochromator.

8. Apparatus as claimed in claim 1 wherein the resonant output signals are signals responsive to molecular emission, including at least one of a fluorescence or phosphorescence signal.

9. Apparatus as claimed in claim 1 wherein the non-resonant signals include at least one of a Rayleigh signal, Mie, Brillouin, and Raman scattering signal.

10. Apparatus as claimed in claim 1 wherein the signal generator generates input signals such that the first and second resonant output signals have substantially the same spectra.

11. Apparatus as claimed in claim 10 wherein the signal generator further comprises means for generating the first and the second input signals in sequence.

12. Apparatus as claimed in claim 1 wherein the means for distinguishing the output signals comprises means for expressing the first and the second resonant output signals as a single eigenvector, wherein the first and the second non-resonant output signals are not capable of being expressed as a single eigenvector.

13. Apparatus as claimed in claim 1 wherein the means for distinguishing the output signals comprises means for selecting a predetermined number of eigenvectors for expressing the first and the second resonant output signals.

14. Apparatus as claimed in claim 1 wherein the distinguishing means comprises means for applying principal components regression analysis to the first second output signals, whereby the resonant output signals are distinguished from the non-resonant output signals.

15. Apparatus as claimed in claim 1 wherein the distinguishing means comprises means for applying multivariate quantitative analysis to the first and second output signals, whereby the first and the second resonant output signals are distinguished from the first and the second non-resonant output signals.

16. Apparatus as claimed in claim 15 wherein the first and second input wavelengths are separated by about 3 nanometers to about 40 nanometers.

17. Apparatus as claimed in claim 14 wherein the first resonant output signal and the second resonant output signal have a first spectrum and a second spectrum respectively, and wherein the first non-resonant output signal and the second non-resonant output signal have a third spectrum and a fourth spectrum respectively, and

wherein the signal generator generates input signals such that the first and the second spectra are substantially the same, and such that the third and the fourth spectra are different, and

wherein the means for principal components regression analysis comprises (i) means for retaining the first and the second resonant output signals having the same spectra and (ii) means for rejecting the first and the second non-resonant output signals having different spectra.

18. Apparatus as claimed in claim 1 wherein the analyte has an absorption level, and wherein the generator has one or more power levels, and wherein the generator further comprises means for selecting the first and the second wavelengths such that the mathematical product of the generator power level and the analyte absorption level is the same at the first and at the second wavelengths respectively.

19. Apparatus as claimed in claim 1 wherein the signal generator generates the first and the second input signals in sequence, and wherein the first and the second non-resonant output signals comprise first and second Raman signals respectively, the first and the second Raman signals having a first and a second Raman spectrum respectively, and

wherein the first Raman spectrum is at a first spectral position dependent on the first wavelength, and the second Raman spectrum is at a second spectral position dependent on the second wavelength, and wherein the first spectral position is different from the second spectral position.

20. Apparatus as claimed in claim 1 wherein the signal generator generates the first and the second input signals in sequence, and wherein the first and the second non-resonant output signals comprise first and second Rayleigh scattering signals respectively, the first and the second Rayleigh signals having a first and a second Rayleigh spectrum respectively, and

wherein the first Rayleigh spectrum is at a first spectral position dependent on the first wavelength, and the second Rayleigh spectrum is at a second spectral position dependent on the second wavelength, and wherein the first spectral position is different from the second spectral position.

21. Apparatus for determining the analyte content of a sample, the apparatus comprising:

(a) a signal generator for generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) a means for directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal, and (ii) a first non-resonant output signal whose peak wavelength is dependent on the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal, and

(c) a detector for detecting the first and second output signals; and

(d) means for distinguishing the first resonant output signal from the second resonant output signal to obtain data about the analyte content of the sample.

22. Apparatus as claimed in claim 21

wherein the first resonant signal has a first spectrum comprising first and second component spectral curves, and wherein the second resonant signal has a second spectrum comprising a third and fourth component spectral curves,

wherein the first and the third component spectral curves are related, and wherein the second and the fourth component spectral curves are related, and

wherein the distinguishing means comprises means for distinguishing the first and the third related spectral curves from the second and the fourth related spectral curves.

23. Apparatus as claimed in claim 21 wherein the resonant output signals are signals responsive to molecular emission, including at least one of a fluorescence or phosphorescence signal.

24. Apparatus as claimed in claim 21 wherein the non-resonant output signals include at least one of a Rayleigh signal, Mie, Brillouin, and Raman scattering signal.

25. Apparatus for determining the analyte content of a sample, the apparatus comprising:

(a) a signal generator for generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) a means for directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal, and (ii) a first non-resonant output signal whose peak wavelength is dependent on the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal, and

(c) a detector for detecting the first and second output signals; and

(d) means for distinguishing the first non-resonant output signal from the second non-resonant output signal to obtain data about the analyte content of the sample.

26. Apparatus as claimed in claim 25 wherein the first non-resonant output signal has a first spectrum and the second non-resonant output signal has a second spectrum, wherein the first and the second spectra are unrelated, and

wherein the distinguishing means comprises means for distinguishing between the unrelated spectra.

27. A method for determining the analyte content of a sample, the method comprising the steps of:

(a) generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal and (ii) a first non-resonant output signal whose peak wavelength is dependent upon the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal;

(c) detecting the first and the second output signals; and

(d) distinguishing the resonant output signals from the non-resonant output signals to obtain data about the analyte content of the sample.

28. The method of claim 27 wherein the step of generating the first and the second input signals comprises:

(i) generating the signals from two separate sources, each one for generating a source signal, and (ii) modulating the source signals for selectively directing the source signals to the sample as input signals.

29. The method of claim 28 wherein the sources comprise laser diodes operating at different wavelengths.

30. The method of claim 28 wherein the step of modulating the source signals comprises modulating the signals using a wavelength modulating device, and wherein the step of directing the source signals comprises guiding the signals via a fiberoptic waveguide having a common waveguide and two limbs, each limb being directed to one of the sources, and the common waveguide being directed to the wavelength modulating device.

31. The method of claim 30 wherein the step of modulating the generated signals comprises modulating the signals by a liquid crystal filter.

32. The method of claim 27 comprising the additional step of filtering the output signals immediately before the step of detecting the output signals.

33. The method of claim 27 wherein the resonant output signals are signals responsive to molecular emission, including at least one of a fluorescence or phosphorescence signal.

34. The method of claim 27 wherein the non-resonant signals include at least one of a Rayleigh signal, Mie, Brillouin, and Raman scattering signal.

35. The method of claim 27 wherein the step of generating the input signals comprises generating the input signals such that the first and second resonant output signals have substantially the same spectra.

36. The method of claim 27 wherein the step of distinguishing the output signals comprises expressing the first and the second resonant output signals as a single eigenvector, wherein the first and the second non-resonant output signals are not capable of being expressed as a single eigenvector.

37. The method of claim 27 wherein the step of distinguishing the output signals comprises selecting a predetermined number of eigenvectors for expressing the first and the second resonant output signals.

38. The method of claim 27 wherein the step of distinguishing the output signals comprises applying principal components regression analysis to the first output and second output signals, whereby the resonant output signals are distinguished from the non-resonant output signals.

39. The method of claim 27 wherein the step of distinguishing the output signals comprises applying a multivariate quantitative analysis to the first and the second output signals, whereby the first and the second resonant output signals are distinguished from the first and the second non-resonant output signals.

40. The method of claim 39 wherein the step of generating the input signals comprises generating the signals such that the first and the second wavelengths are separated by about 3 nanometers to about 40 nanometers.

41. The method of claim 38 wherein the first and the second resonant output signals have a first and a second spectrum respectively, and wherein the first and the second non-resonant output signals have a third and a fourth spectrum respectively, and

wherein the step of generating the input signals comprises selecting the first and the second wavelengths such that the first and the second spectra are substantially the same, and such that the third and the fourth spectra are different, and

wherein the step of applying principal components regression analysis comprises (i) retaining the first and the second resonant output signals having the same spectra and (ii) rejecting the first and the second non-resonant output signals having different spectra.

42. The method of claim 27 wherein the step of generating the input signals comprises generating the first and the second input signals in sequence, and wherein the first and the second non-resonant output signals comprise first and second Raman signals respectively, the first and the second Raman signals having a first and a second Raman spectrum respectively, and

wherein the first Raman spectrum is at a first spectral position dependent on the first wavelength, and the second Raman spectrum is at a second spectral position dependent on the second wavelength, and wherein the first spectral position is different from the second spectral position.

43. The method of claim 27 wherein the step of generating the input signals comprises generating the first and the second input signals in sequence, and wherein the first and the second non-resonant output signals comprise first and second Rayleigh scattering signals respectively, the first and the second Rayleigh signals having a first and a second Rayleigh spectrum respectively, and

wherein the first Rayleigh spectrum is at a first spectral position dependent on the first wavelength, and the second Rayleigh spectrum is at a second spectral position dependent on the second wavelength, and wherein the first spectral position is different from the second spectral position.

44. A method for determining the analyte content of a sample, the method comprising the steps of:

(a) generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal, and (ii) a first non-resonant output signal whose peak wavelength is dependent on the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal, and

(c) detecting the first and second output signals; and

(d) distinguishing the first resonant output signal from the second resonant output signal to obtain data about the analyte content of the sample.

45. The method of claim 44 wherein the first resonant output signal has a first spectrum comprising first and second component spectral curves, and wherein the second resonant output signal has a second spectrum comprising third and fourth component spectral curves,

wherein the first and the third component spectral curves are related, and wherein the second and the fourth component spectral curves are related, and

wherein the step of distinguishing the output signals comprises distinguishing the first and the third related spectral curves from the second and the fourth related spectral curves.

46. The method of claim 44 wherein the resonant output signals are signals responsive to molecular emission, including at least one of a fluorescence or phosphorescence signal.

47. The method of claim 44 wherein the non-resonant signals include at least one of a Rayleigh signal, Mie, Brillouin, and Raman scattering signal.

48. A method for determining the analyte content of a sample, the method comprising the steps of:

(a) generating a first input signal having a first wavelength, and a second input signal having a second wavelength, the two wavelengths differing by at least about 3 nanometers;

(b) directing the first and second input signals to a sample containing an analyte to generate, respectively, a first output signal and a second output signal due to interactions between the input signals and the analyte,

wherein the first output signal comprises;

(i) a first resonant output signal whose peak wavelength is substantially independent of the wavelength of the first input signal, and (ii) a first non-resonant output signal whose peak wavelength is dependent on the wavelength of the first input signal, and

wherein the second output signal comprises;

(i) a second resonant output signal whose peak wavelength is substantially independent of the wavelength of the second input signal, and (ii) a second non-resonant output signal whose peak wavelength is dependent on the wavelength of the second input signal, and

(c) detecting the first and second output signals; and

(d) distinguishing the first non-resonant output signal from the second non-resonant output signal to obtain data about the analyte content of the sample.

49. The method of claim 48 wherein the first non-resonant signal has a first spectrum and the second non-resonant signal has a second spectrum, wherein the first and the second spectra are unrelated, and

wherein the step of distinguishing the output signals comprises distinguishing between the unrelated spectra.