CAPILLARY FLOWCELL FOR MULTIPLE WAVELENGTH DETECTION

Abstract: A capillary flowcell (12) includes an optical axis, the optical axis being defined by, in sequential order, (a) a first (entrance) aperture (14), (b) a first lens (26), (c) a capillary tube (16) having its long axis centered across the optical axis, and (d) a second lens (28). The first lens (26) focuses electromagnetic radiation from the entrance aperture (14), passes through the inner diameter of capillary tube (16) thus maximizing the signal generated by the analyte within the capillary tube (16). The second lens (28) focuses electromagnetic radiation which has passed through the capillary tube (16).

Description: absorbed by the analyte(s) in the flowcell is transmitted through the flowcell, and is converted to an electrical signal by a detector element. If the detector element is sufficiently sensitive and of sufficient area to intercept the transmitted optical beam, it is not necessary to refocus the beam. Variable wavelength detectors of the prior art (which determine a single wavelength of electromagnetic radiation, sλ), are shown in Figure 1 and in Figure 2. Figure 1 shows a diagrammatic representation of the path of light through a capillary flowcell in a variable wavelength detector. Monochromatic light at a single wavelength (sλ) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 by passing through a mask 13 at the entrance aperture 14. The mask 13 is placed to restrict optical radiation which would not pass through the inner diameter of the capillary (and thus the analyte) within the flowcell. The electromagnetic radiation passes through the capillary tube wall 16 and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which has passed through the analyte 18 and which then passes through the exit aperture 20 is converted to an electrical signal by a detector element 22. The optical axis α of the flowcell system is shown for reference purposes. Figure 2 shows a diagrammatic representation of the path of light through an alternate capillary flowcell in a variable wavelength detector. Monochromatic light at a single wavelength (sλ) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 at the entrance aperture 14. A spherical ball 1 Capillary Flowcell For Multiple Wavelength Detection Technical Field This invention relates to an improved capillary flowcell apparatus for measuring absorbance of radiation, especially optical radiation, in a multiple wavelength detector (MWD). Background of the Invention Optical absorbance across a capillary flowcell is used to determine the presence and/or concentration of an analyte within the capillary. The basic optical configuration of a multiple wavelength detector is different from that of a variable wavelength detector. However, each type of detector is often configured as a dual beam design, with a flowcell containing analyte(s) in one beam, and a reference flowcell in an alternate reference beam. Such designs permit the determination of lower concentrations of analyte by compensating, for example, for fluctuations in source energy, and reduction of signal variation as a function of wavelength. Electromagnetic radiation which is delivered through the analyte 16 and to the detector 22 (i.e., the signal) competes with electromagnetic radiation which is delivered to the detector 22 without having passed through the analyte 16 (i.e., "noise"). In a variable wavelength detector, broad-band optical radiation from a source is passed through a wavelength selection device such as a monochromatoi * . A narrow, nearly monochromatic band of electromagnetic radiation is selected and focused upon the entrance aperture of the flowcell. Optical radiation which is not scattered or lens 15 is then used to reimage the entrance aperture 14 onto the inner diameter of the capillary tube 16 containing the analyte 18. The focused electromagnetic radiation passes through the capillary tube 16 wall and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which has passed through the analyte 18 and which passes through the exit aperture 20 is converted to an electrical signal by a detector element 22. The optical axis α of the flowcell system is shown for reference purposes. In a multiple wavelength detector, a source of optical radiation including a multiplicity of wavelengths is focused onto a flowcell. The analyte(s) in the flowcell may absorb optical radiation at a multiplicity of different wavelengths. Optical radiation which is not absorbed by the analyte(s) in the flowcell is transmitted through the flowcell, and through a polychromator. The polychromator disperses the transmitted optical radiation into many narrow bands, as a function of band wavelength. Each narrow band is focused onto a different detector element in a detector array, and is converted to an electrical signal. The absorbance of the analyte(s) in the flowcell is thus measured as a function of wavelength absorption or transmission of many different wavelengths. A multiple wavelength detector such as that found in the prior art, having a beam of multiple wavelength of electromagnetic radiation (mλ), is shown in Figure 3. Representative wavelengths (λi, λ2, and λ3) are shown after separation by the polychromator. Figure 3 shows a diagrammatic representation of the path of light through a capillary flowcell in a multiple wavelength detector. Polychromatic light at various wavelengths ( λ) is emitted from an electromagnetic radiation source 10. Electromagnetic radiation (shown as arrows) enters the flowcell 12 by passing through a mask 13 at the entrance aperture 14. The mask 13 is placed to restrict optical radiation which would not pass through the inner diameter of the capillary (and thus the analyte 18) within the flowcell. The electromagnetic radiation passes through the capillary tube wall 16 and through the analyte 18 contained within the capillary tube. Electromagnetic radiation which, has passed through the analyte 18 and which passes through the exit aperture 20 is split into discrete bands CΛi, λ2 and λ3) by the polychromator 24. As shown, the polychromator can include an entrance aperture 21. Alternatively, the exit aperture 20 of the flowcell 10 can act as the entrance aperture for the polychromator. Each discrete wavelength of electromagnetic radiation is then converted to an electrical signal by discrete detector elements 22. The optical axis α of the flowcell system is shown for reference purposes. The fundamental design differences between variable wavelength detectors and multiple wavelength detectors place conflicting demands upon flowcell design, especially with respect to optical radiation transmitted through the analyte. Variable wavelength detectors are often designed such that the beam of optical radiation does not require focusing after passing through the analyte. Rather, the beam is uninterrupted between the capillary containing the analyte and the detector element. Multiple wavelength detectors generally require that the beam be refocused for the polychromator after the beam has passed through the analyte sample. The exit aperture of the flowcell can serve as the entrance aperture for the polychromator. The presence of a capillary column within the flowcell restricts flowcell design. Because chromatic integrity is maintained by minimizing off-column chromatic dispersion, on-column detection is usual. In on- column detection, the capillary forms part of the flowcell. Albin et al., "Fluorescence Detection in Capillary Electrophoresis: Evaluation of Derivatizing Reagents and Techniques", Anal. Chem. 63 (1991) describe a capillary electrophoresis fluorescence detector. A single sapphire lens is used to focus the monochromatic beam before the beam is passed through the capillary flowcell. Kobayashi et al. ("Photodiode Array Detection in High-Performance Capillary Electrophoresis", J. Chrom. 480 (1989):179-184) describe replacement of a standard cell of a spectrometer with a capillary. The light intensity of the apparatus was decreased to prevent saturation of the diode array. Moring et al., "Analytical Aspects of an Automated Capillary Electrophoresis System", LC:GC 8(l):34-46 (1990) describes a variable wavelength detector in which one sapphire lens is used to focus the monochromatic light prior to the time the beam is focused on the capillary flowcell. A capillary flowcell is described in Sepaniak et al., "Instrumental Developments in Micellar Electrokinetic Capillary Chromatography' ' , J. Chrom. 480 (1989):185-196. The capillary flowcell is produced by removing a portion of the capillary coating, epoxyin the capillary between two microscope slides, attaching the capillary and slides to a bracket, painting the front face of the apparatus black, and using an argon laser to etch the painted section. ' The flowcell is completed by masking all but a small portion with electrical tape. The flowcell is described for use with each of a variable wavelength detector and a multiple wavelength detector. When the flowcell is used in a multiple wavelength detector, a beam of optical radiation originates in a deuterium lamp, passes first through the capillary, and then through two separate lenses before it is focused at the entrance of the spectrometer. Summary of the Invention A capillary flowcell of this invention comprises a holding means including an optical axis, the optical axis being defined by, in sequential order, (a) a first (entrance) aperture, (b) a first lens, (c) a capillary tube having its long axis centered across the optical axis, and (d) a second lens. The first lens focuses electromagnetic radiation from the entrance aperture to pass through the inner diameter of the capillary tube, thus maximizing the signal generated by the analyte within the capillary tube. The second lens focuses electromagnetic radiation which has passed through the capillary tube. Generally, the second lens focuses the electromagnetic radiation through the exit aperture of the flowcell or through the entrance aperture of a polychromator, thus maximizing the "signal" and minimizing the amount of light which is scattered to produce "noise". More preferably, the entrance aperture of the polychromator is the exit aperture of the flow cell. The holding means preferably includes a means for holding a capillary tube precisely, so that the center of the capillary tube is held directly across the optical axis of the holding means. The holding means can conveniently comprise two mating parts, the union of the parts providing a channel through which the capillary tube is held. Brief Description of the Drawings Figure 1 shows a diagrammatic view of a variable wavelength detector and capillary flowcell of the prior art. Figure 2 shows a diagrammatic view of an alternate variable wavelength detector and capillary flowcell of the prior art. Figure 3 shows a diagrammatic view of a multiple wavelength detector and capillary flowcell of the prior art. Figure 4 shows a diagrammatic view of a variable wavelength detector and capillary flowcell of this invention. Figure 5 shows a cross-sectional view of a capillary flowcell of this invention. Figure 6 shows the mating surface of one subunit of the flowcell of Figure 5. Disclosure of the Invention Including Best Mode The subject invention is a flowcell design which provides enhanced imaging capability for flowcell function with a polychromator in a multiple wavelength detector. It provides increased throughput to allow lower limits of detection of analytes in a capillary; a means of locating and holding the capillary in place while allowing easy replacement of the capillary; and the ability to substitute for a typical liquid chromatography flowcell in a standard multiple wavelength detector. Similar numbers refer to similar function throughout the Figures. The figures are drawn for clarity and are not drawn to scale. Figure 4 shows a diagrammatic representation of the path of a light beam through a capillary flowcell of this invention. Polychromatic light at various wavelengths (mλ) is emitted from an electromagnetic radiation source 10. Convergent incident electromagnetic radiation (shown as arrows) enters the flowcell 12 at the entrance aperture 14. The electromagnetic radiation passes through a first lens 26. The first lens 26 focuses the electromagnetic radiation entering the flowcell at the entrance aperture onto the analyte 18 contained within the inner diameter of the capillary tube 16. This focusing causes the electromagnetic radiation to pass substantially through the analyte 18 contained within the inner diameter of the capillary tube 16. Such focusing of the electromagnetic radiation beam maximizes the amount of electromagnetic radiation which passes through the analyte, and thus maximizes the "signal". It also minimizes the amount of electrical radiation which does not pass through the analyte and appears as "noise". Electromagnetic radiation which has passed through the analyte 18 then passes through the second lens 28. The second lens focuses the "signal" to pass through the exit aperture 20 and/or the entrance aperture 21 of the polychromator 24 while minimizing the "noise" which is transmitted. Preferably, the illuminated area of the analyte is reimaged by the second lens onto the entrance aperture of the polychromator. In a preferred embodiment, the convergent exit angle (ø) of the flowcell matches the entrance angle of the polychromator. The polychromator 24 splits the beam into discrete bands (shown as λi, λ2 and λβ). As with the previous embodiments, each discrete wavelength of electromagnetic radiation is then converted to an electrical signal by discrete detector elements 22. As shown in Figure 5, the capillary flowcell 101 includes a holding means 103 having a bore 119 therethrough. A beam of electromagnetic radiation is focused and refocused by optical elements within the bore 119. A narrow beam of multiple wavelength electromagnetic radiation is preferentially transmitted along the optical axis α by the selective selection, focusing and reimaging of the optical elements. A source of incident multiple wavelength electromagnetic radiation (not shown), -typically a deuterium lamp or other source, is focused into a beam which includes multiple wavelengths of electromagnetic radiation. Preferably the beam is a convergent beam which is focused to pass through SUBSTITUTE SHEET the entrance aperture, and which thereafter impinges completely upon the first lens. The optical axis α is a line which is generally defined as the center of the path of multiple wavelength electromagnetic radiation as it is shaped by, in sequential order: (a) an entrance aperture 105; (b) a first lens 107; (c) a capillary tube 109 having its long axis centered across the optical axis α; and (d) a second lens 111. The first lens 107 focuses electromagnetic radiation incident from the aperture 105 to pass substantially through the inner diameter of the capillary tube 109. The second lens 111 focuses electromagnetic radiation which has passed through the capillary tube along the optical axis α. The second lens 111 focuses electromagnetic radiation to pass through the exit aperture 113, if such an aperture is present, and/or focuses the radiation onto the entrance aperture of a polychromator. It is preferable that the convergent angle of the beam as it exits the flowcell matches the acceptance angle of the polychromator. The exit aperture 113 of the flowcell and the entrance aperture of the polychromator (not shown) can be the same port.. The diameter of the incident beam is first limited and shaped by the entrance aperture 105. As shown, each of the entrance aperture 105 and the exit aperture 113 is an opening within an aperture plate 115 and 127, respectively. In a preferred embodiment, the aperture plates 115 and 127 are held in place at a recessed surface of the holding means 103 by a retaining ring 117. This recessed aperture configuration acts to protect the aperture plate 115 and 127 while permitting the easy replacement of the aperture plate 115 and 127 if an aperture of different size is desired. SUBSTITUTE SHEET For ease of construction the aperture plate 115 and 127 can be made of plastic. Preferably, the diameter of the entrance aperture 105 is chosen to correspond to the maximum diameter of the image of the aperture 105 that will fit onto the inner diameter of the capillary 109. Preferably, the diameter of the exit aperture 113 is chosen to correspond to the maximum diameter of the image of the inner diameter of the capillary tube which will fit onto the entrance aperture of the polychromator. As it passes through the entrance aperture 105, the beam enters a bore 119 within the holding means 103. Situated within this bore 119 are, in sequential order, a first lens 107, a capillary tube 109 having its long axis centered across the optical axis, and a second lens 111. The first lens 107 and the second lens 111 are preferably spherical sapphire lenses. Sapphire lenses are preferred due to their ability to efficiently transmit electromagnetic radiation over the wavelength range of interest, their high index of refraction and ability to focus electromagnetic radiation over short focal lengths, and their reasonable cost. The lenses are generally positioned by manufacturing the bore 119 somewhat smaller than the diameter of the lens. For example, a bore having a diameter of 1.95 mm would be formed to house a lens having a diameter of 2.0 mm. The spherical ball lens is then pressed into position within the bore 119. For this reason, a relatively soft material is preferably used for the holding means 103. Plastic such as Acetal is appropriate for use. SUBSTITUTE SHEET The first lens 107 is placed at an appropriate distance from each of the entrance aperture 105 and the capillary tube 109 so that light which enters the flowcell through the entrance aperture 105 is focused by the first lens to pass through the inner diameter of the capillary tube 109. The capillary tube 109 is preferably a standard capillary tube. Such capillary tubes have an outside diameter (O.D.) of approximately 375 μm, and an inner diameter (I.D.) of approximately 50μm. In one preferred embodiment, when a standard capillary tube is used, and the first lens 107 and second lens 111 are each a sapphire spherical ball lens having a diameter of 2.0 mm. The entrance aperture. 105 is typically 0.5 to 0.6 m in diameter, and is placed approximately 7 mm from the first lens. The capillary tube 109 is preferably placed so that it touches each of the first lens 107 and the second lens 111. The exit aperture 113 is preferably located approximately 7 mm from the second lens 111. The second lens 111 is placed at an appropriate distance from each of the capillary tube 109 and the exit aperture 113 so that light which is incident upon the second lens 111 is focused to pass through the exit aperture 113. In a preferred embodiment, as shown, the holding means includes two mating parts, 103a and 103b. A portion of the outer diameter of the first part 103a is preferably recessed so that the improved flowcell can be retained by a- circular clamping hole in a standard multiple wavelength absorbance detector. The first part 103a includes the entrance aperture 105. Preferably, the entrance aperture 105 is located at the focal point of a SUBSTITUTE SHEET convergent beam produced by the multiple wavelength electromagnetic radiation source. The first part 103a is configured to hold the first lens 107, preferably using a friction or pressure fit. The second part 103b is configured for holding the second lens 111 by a similar means. The second part 103b preferably includes an exit aperture 113. Light which passes through the exit aperture 113 enters a polychromator where the beam is split by wavelength, and the presence or absence of signal at the various wavelengths is determined. In a preferred embodiment, the diameter of the holding means is approximately 2 cm, and the length of the holding , means is approximately 1.3 cm. The first part 103a of the holding means 103 includes a recessed area which mates with a boss of the second part 103b. The two parts are positioned so that a hole 125 in each part aligns for entry of a screw (not shown). The screw maintains the holding means 103 as an integral unit. The capillary tube 109 is positioned so that the long axis of the tube is centered on the optic axis of the system. Figure 6 shows the mating (inner) face of the first part 103a of the holding means 103. As shown m Figure 5. the capillary tube 109 is held in position by a groove 121 in the first part 103a of the holding means 103. The groove 121 can have any desired cross- section, and is conveniently trapezoidal. The groove 121 substantially encloses the capillary tube 109. An O-ring 123 is held within a circular recessed trough on the mating face of one subunit. This O-ring 123 acts to help mate the two subunits, and to hold the capillary tube securely in place with a gentle pressure. The second subunit (not shown) mates with the first subunit to complete the holder apparatus, and the two subunits are SUBSTITUTE SHEET conveniently held together using a pressure -fit secured with a screw or the like. A threaded bore 125 for a screw is shown. While the invention has been described in connection with specific embodiments thereof, those skilled in the art will recognize that various modifications are possible within the principles described herein. Such modifications.- variations, uses, or adaptations of the invention, including such departures from the present disclosure as come within known or customary practice in the art, fall within the scope of the invention and of the appended claims. SUBSTITUTE SHEET

Claims: I Claim: 1. A capillary flowcell for determination of an analyte using multiple wavelength electromagnetic radiation, said flowcell comprising, in sequential order along an optical axis: (a) a first barrier defining a first aperture on said optical axis; (b) a first lens; (c) a capillary tube having its long axis centered across the optical axis; and (d) a second lens; and (e) a holder that holds each of elements (a) - (d) in fixed alignment; wherein the first lens focusses multiple wavelength electromagnetic radiation incident from the first aperture to pass through an inner diameter of the capillary tube; and wherein the second lens focusses electromagnetic radiation which has passed through the capillary tube. 2. A flowcell as in claim 1 wherein said first lens focusses the multiple wavelength electromagnetic radiation incident from said first aperture such that this radiation passes through an inner diameter of the capillary within which sample fluid passes; and wherein the second lens focusses multiple wavelength electromagnetic radiation to pass through a second aperture in a second barrier. 3. A flowcell ' as in claim 2 wherein said second aperture is the entrance aperture of a polychromator. 4. A flowcell as in claim 1 wherein at least one of said first and second lenses is a spherical ball lens. SUBSTITUTE SHEET 5. A flowcell as in claim 4 wherein at least one of the first and second lenses is sapphire. 6. A flowcell as in claim 1 wherein the capillary tube is replaceable. 1. A flowcell comprising: (a) a housing having a bore therethrough, wherein a central axis of said bore defines an optical axis; and positioned along said optical axis, in sequential order, (b) a first lens; (c) a holder for holding a capillary tube such that the optical axis passes through a center of this tube; and (d) a second lens. 8. A flowcell as in claim 7 wherein the first lens focusses incident electromagnetic radiation onto the optical axis within the acceptance angle of the second lens and wherein the second lens focusses incident electromagnetic radiation from the first lens to a point further along the optical axis. 9. A flowcell as in claim 7 wherein the following elements are aligned along the optical axis in the following sequential order: (aO) a mask defining a first aperture that is substantially centered on said optical axis; (b) said first lens; (c) said capillary tube holder such that a channel in this holder is adapted to hold said capillary such that the optical axis passes laterally through the capillary along a path that intersects said capillary substantially at a central axis of said capillary; (d) said second lens; and (e) a second aperture that is substantially centered on said optical axis. SUBSTITUTE SHEET 10. A flowcell as in claim 7 wherein at least one of the first and second lenses is spherical. 11. A flowcell as in claim 7 wherein at least one of said first and second lenses is sapphire. 12. A flowcell as in claim 7 wherein the capillary tube is removable. 13. A flowcell as in claim 7, further comprising a variable wavelength detector aligned to receive light that has passed through a central bore of said capillary tube. 14. A flowcell as in claim 7, further comprising a multiple wavelength detector aligned to receive light that has passed through a central bore of said capillary tube. 15. A method of determining an analyte in a central bore of a capillary tube, said method comprising: (a) directing a beam of multiple wavelength radiation through a first lens that focusses this beam through the analyte in the capillary tube; and (b) focussing the beam after passing through said analyte, with a second lens located collinearly along an optical path that passes through the first lens, through the central bore of the capillary tube and through the second lens, said second lens being positioned to image the beam onto an entrance of a polychro- mator. 16. A method as in claim 15 wherein at least one of said first and second lenses is spherical. 17. A method as in claim 15 wherein at least one of the first and second lenses s sapphire. SUBSTITUTE SHEET

Inventors: SAPP EDWIN, SAPP, EDWIN
Applicants: APPLIED BIOSYSTEMS, APPLIED BIOSYSTEMS, INC.
Application year: 1992
Main classification: G01N21
IPC codes: G01N21, G01N27, G01N30