COMBINED FLUORESCENCE AND ABSORPTION DETECTOR FOR ON-COLUMN DETECTION AFTER CAPILLARY SEPARATION TECHNIQUES

Abstract

A system and method for performing UV LED-based absorption detection for capillary liquid chromatography for detecting and quantifying compounds in a liquid, wherein a simplified system eliminates the need for a beam splitter and a reference cell by using a stable UV source, and power requirements are reduced, resulting in a portable and substantially smaller system with relatively low detection limits.

Claims

1. A combined fluorescence detector and absorption detector system for on-column detection after using capillary separation techniques, said system comprising: an LED for generating a UV light; a first lens for focusing the UV light from the LED; a dichroic mirror for reflecting the UV light in a first direction; a second lens for focusing the UV light in the first direction; a capillary column for receiving the UV light to thereby enable on-column detection; an absorption detector for receiving the UV light that has passed through the capillary column and performing absorption detection; the dichroic mirror now passing light that is emitted by fluorescence from one or more compounds within the capillary column, the fluorescence light moving in a second direction opposite the first direction; a third lens for focusing the fluorescence light in the second direction; and a fluorescence detector for receiving the fluorescence light and performing fluorescence detection.

2. The system as defined in claim 1 wherein the system further comprises an excitation filter disposed between the first lens and the dichroic mirror for filtering the UV light.

3. The system as defined in claim 1 wherein the system further comprises at least one slit for passing the UV light received from the second lens in the first direction and disposed for reducing stray light from entering the capillary column.

4. The system as defined in claim 3 wherein the system further comprises an emission filter disposed between the dichroic mirror and the third lens for filtering the fluorescence light.

5. The system as defined in claim 4 wherein the system further comprises at least one slit or pinhole for passing the fluorescence light received from the third lens in the second direction and disposed for reducing stray light from passing through.

6. The system as defined in claim 5 wherein the absorption detector is further comprised of a system for analyzing absorption of the UV light by at least one compound disposed within the capillary column by analyzing the UV light that is received by the absorption detector.

7. The absorption detector as defined in claim 6 wherein the absorption detector is further comprised of: a photodiode for receiving the UV light from the capillary column; an operational amplifier for receiving current from the photodiode and converting it into voltage values; and an analog-to-digital converter for receiving the voltage values from the operational amplifier and converting it into digital values.

8. A method for combining fluorescence detection and absorption detection in a detection system for performing on-column capillary detection after using capillary separation techniques, said method comprising the steps of: providing a light emitting diode (LED), a first lens for receiving and focusing the UV light from the LED, a dichroic mirror for reflecting the UV light in a first direction, a second lens for focusing the UV light in the first direction, at least one slit for passing the UV light received from the second lens in the first direction, a capillary column for receiving the UV light, and an absorption detector for receiving the UV light that has passed through the capillary column and performing absorption detection; providing the dichroic mirror that is passing light that is emitted by fluorescence from one or more compounds within the capillary column, the fluorescence light moving in a second direction opposite the first direction, a third lens for focusing the fluorescence light in the second direction, and a fluorescence detector for receiving the fluorescence light and performing fluorescence detection; generating the UV light from the LED; measuring the UV light that passes through the capillary column by using the absorption detector; analyzing absorption of the UV light by the at least one compound within the capillary column by analyzing the UV light that is received by the detector; passing the fluorescence light from the capillary column through the dichroic mirror; measuring the fluorescence light from the at least one compound in the capillary column; and analyzing absorption of the fluorescence light by the at least one compound within the capillary column by analyzing the fluorescence light that is received by the fluorescence detector.

9. The method as defined in claim 8 wherein the method further comprises: disposing an excitation filter between the first lens and the dichroic mirror; and filtering the UV light from the first lens.

10. The method as defined in claim 9 wherein the method further comprises providing at least one slit for passing the UV light received from the second lens in the first direction; and reducing stray light from entering the capillary column.

11. The method as defined in claim 10 wherein the method further comprises: disposing an emission filter between the dichroic mirror and the third lens; and filtering the fluorescence light from the dichroic mirror.

12. The method as defined in claim 11 wherein the method further comprises: providing at least one slit or pinhole for passing the fluorescence light received from the third lens in the second direction; and reducing stray light from passing through at least one slit or pinhole.

13. The method as defined in claim 12 wherein the method further comprises providing a system for analyzing absorption of the UV light by the at least one compound within the capillary column by analyzing the UV light that is received by the absorption detector.

14. The method as defined in claim 8 wherein the method further comprises increasing the amount of the UV light received by the detector by at least two orders of magnitude.

15. The method as defined in claim 8 wherein the method further comprises: selecting a wavelength of the UV light generated by the LED; and selecting the excitation filter to match the wavelength of the UV light generated by the LED to thereby reduce stray light from reaching the capillary column.

16. The method as defined in claim 8 wherein the method further comprises positioning the second lens relative to the dichroic mirror such that a focal point of the UV light from the second lens is equal to or less than an inside diameter (ID) of the capillary column.

17. The method as defined in claim 6 wherein the method further comprises: providing a photodiode in the absorption detector, providing an operational amplifier for receiving current from the photodiode and converting it into voltage values and providing an analog-to-digital converter for receiving the voltage values from the operational amplifier. receiving the UV light from the capillary column at the photodiode; converting the UV light into voltage values using the operational amplifier; and converting the voltage values into a digital signal.

18. The method as defined in claim 10 wherein the method further comprises the step of providing power to the LED, the absorption detector and the fluorescence detector using a DC power source to thereby enable the detection system to be portable.

19. A combined fluorescence detector and absorption detector system for on-column detection after using capillary separation techniques, said system comprising: an LED for generating a UV light; a system of lenses, a mirror and a slit for directing and focusing the UV light; a capillary column for receiving the UV light to thereby enable on-column detection; an absorption detector for receiving the UV light that has passed through the capillary column and performing absorption detection; the dichroic mirror also passing light that is emitted by fluorescence from one or more compounds within the capillary column, the fluorescence light moving in a second direction opposite the first direction; a third lens and a slit for directing and focusing the fluorescence light in the second direction; and a fluorescence detector for receiving the fluorescence light and performing fluorescence detection.

20. A method for combining fluorescence detection and absorption detection in a detection system for performing on-column capillary detection after using capillary separation techniques, said method comprising the steps of: providing a UV light source, a system of lenses, a dichroic and a slit for directing the UV light in a first direction, a capillary column for receiving the UV light, and an absorption detector for receiving the UV light that has passed through the capillary column and performing absorption detection; providing the dichroic mirror that is passing light that is emitted by fluorescence from one or more compounds within the capillary column, the fluorescence light moving in a second direction opposite the first direction, providing a third lens and a slit for directing and focusing the fluorescence light in the second direction, and a fluorescence detector for receiving the fluorescence light and performing fluorescence detection; generating the UV light from the LED; measuring the UV light that passes through the capillary column by using the absorption detector; analyzing absorption of the UV light by the at least one compound within the capillary column by analyzing the UV light that is received by the detector; passing the fluorescence light from the capillary column through the dichroic mirror; measuring the fluorescence light from the at least one compound in the capillary column; and analyzing absorption of the fluorescence light by the at least one compound within the capillary column by analyzing the fluorescence light that is received by the fluorescence detector.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0055] FIG. 1 is a first diagram of a first prior art system that may be modified to function as part of the present invention and shows the major hardware elements of a capillary LC system.

[0056] FIG. 2 is a second diagram of the system of FIG. 1 showing more construction detail of a capillary LC system.

[0057] FIG. 3 is a diagram of the components in a modified system of FIG. 1.

[0058] FIG. 4 is a diagram of the components in a different modified system of FIG. 1.

[0059] FIG. 5 is a diagram of a first embodiment of the invention.

DETAILED DESCRIPTION

[0060] Reference will now be made to the drawings in which the various embodiments of the present invention will be given numerical designations and in which the embodiments will be discussed so as to enable one skilled in the art to make and use the invention. It is to be understood that the following description illustrates embodiments of the present invention and should not be viewed as narrowing the claims which follow.

[0061] FIG. 5 is a diagram of a first embodiment of the invention. This first embodiment combines fluorescence and spectrophotometric (absorption) detection in a single compact system that is suitable for on-column detection with capillary column-based separations.

[0062] There are two detection channels in the system. The first detection channel is an absorption channel that includes all of the elements in a path of UV light from a light-emitting diode (LED) light source to an absorption detector. An absorption channel is based on the design shown in the prior art. However, the absorption channel may be modified in order for a second detection channel to be added to the system.

[0063] The second detection channel is a fluorescence channel that includes all of the elements in a path of UV light from the LED light source to a fluorescence detector.

[0064] Detection of the at least one compound in the capillary column is based on on-column absorbance at over a selectable wavelength band, depending on the combination of LED selection and bandpass filter placed between the LED and the capillary column.

[0065] Adding the fluorescence channel to the prior art absorption detector incorporates several unique features that were not part of any previous dual detector designs. Before these unique features are identified, the elements of the two detection channels will be described.

[0066] The elements of the first embodiment that are the same as the elements of the prior art may also have the same features and characteristics as described above. The elements of the system in FIG. 5 include a UV-based LED 60, a first ball lens 62, and an excitation (band-pass) filter 64 that may be tuned to the LED 60 light source. A new feature of the first embodiment is necessary in order to provide the two detection channels. The new feature is a dichroic mirror 66. It may also be referred to as a long-pass dichroic mirror 66 or dichroic beam splitter as understood by those skilled in the art. The dichroic mirror 66 enables the first embodiment to have the dual detector channels operate simultaneously.

[0067] The dichroic mirror 66 substantially reflects all of the UV light from the UV-based LED 60 in a first direction that may be at approximately a right angle to the path traveled from the source LED 60. The absorption channel light path is indicated by the dotted lines 68.

[0068] The UV light is reflected off the dichroic mirror towards a second lens 70. The second lens 70 may be a convex lens in order to focus the UV light.

[0069] After the second lens 70 may be a slit 72 that may be comprised of razor blades, a capillary column 74 that may have an inner diameter (ID) of approximately 150 m and an outer diameter of approximately 365 m, and a silicon photodiode detector 76. That description completes all of the elements of the absorption channel in the system.

[0070] The fluorescence channel begins using the same UV-based LED 60, the first ball lens 62, and the excitation filter 64 that may be tuned to the LED 60 light source. The dichroic mirror 66 is also used to send the UV light in a first direction 78 through the second lens 70, the slit 72 and into the capillary column 74.

[0071] However, at this point the path of light diverges. One or more compounds in the capillary column 72 may fluoresce and give off a light that will be referred to hereinafter as fluorescence light. The fluorescence light that is being measured travels in the opposite direction to the first direction 78 as a second direction 80.

[0072] The fluorescence light is focused by the second lens 70 as shown by lines 82 so that it passes through the dichroic mirror 66. An emission filter 84 may filter the fluorescence light before it passes through a third lens 86 which focuses the fluorescence light through a slit or pinhole 88 and into a fluorescence detector 90.

[0073] Some of the unique features of the first embodiment include the same UV LED 60 is used for both detection channels. The desirable properties of the LED 60 are compact size, low power consumption, high spectral irradiance, low cost, and stable output power. It is the stable output of the LED 60 that enables the absorption part of the system to operate without a reference channel. That same stability may also be essential for low-noise fluorescence detection because the fluorescence signal depends directly on the radiant flux hitting the sample.

[0074] Another features is that the second lens 70 that focuses the light from the LED 60 into the capillary column 74 is also used as the primary collection optic for the fluorescence detector 90. An epi illumination scheme ensures that the addition of the fluorescence channel to the absorption detector 76 does not in any way degrade the performance of the absorption detector.

[0075] A third feature of the first embodiment is that the incoming excitation radiation from the LED 60 and the outgoing fluorescence light from the at least one compound in the capillary column 74 are separated by the dichroic mirror 66 which also functions as a beam splitter may reflect the excitation wavelengths of the LED 60 and reflect the fluorescence wavelengths of the fluorescence light.

[0076] The combining of the two optical paths 68, 82 of the absorption channel and the fluorescence channel ensures that both detectors 76, 90 are responding to the same small volume in the capillary column 74.

[0077] A fourth feature is that after the two light paths 68, 82 are separated, one or more optical filters 84 may be placed in the fluorescence emission path 82 to discriminate against residual excitation light and to add a degree of specificity for selected classes of compounds in the capillary column 74. The wavelength difference between the excitation radiation from the LED 60 and the peak of the fluorescence signal depends on the electronic structure of the fluorescing molecule, so different excitation wavelength and emission filter combinations may be used to target specific classes of compounds.

[0078] In an alternative embodiment of the invention, the emission filter 84 and fluorescence detector 90 in the fluorescence emission path 82 may be replaced by a compact spectrometer that may record an entire fluorescence spectrum.

[0079] It is observed that both the emission channel and the absorption channel may be monitored simultaneously and continuously. Each channel may provide a measure of analyte concentration, with differing sensitivities, depending on the electronic structure of the analyte molecule. The ratio of the fluorescence to the absorbance is, to a first approximation, independent of concentration. Rather it may be a measure of fluorescence quantum yield, and it may provide a molecular signature that is not available through either detection channel by itself. In the first embodiment of the invention that incorporates a spectrometer in the emission channel, the recorded spectra may provide information about eluting analytes that aids in analyte identification.

[0080] Although only a few example embodiments have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the example embodiments without materially departing from this invention. Accordingly, all such modifications are intended to be included within the scope of this disclosure as defined in the following claims. It is the express intention of the applicant not to invoke 35 U.S.C. 112, paragraph 6 for any limitations of any of the claims herein, except for those in which the claim expressly uses the words means for together with an associated function.