WO2002042751A1

WO2002042751A1 - Flow through cell for spectrofluorometry

Info

Publication number: WO2002042751A1
Application number: PCT/AU2001/001363
Authority: WO
Inventors: Robert William Blair
Original assignee: Varian Australia Pty Ltd
Priority date: 2000-11-21
Filing date: 2001-10-24
Publication date: 2002-05-30
Also published as: AU2002210277A1; AUPR161000A0

Abstract

A flow through cell for use in spectrofluorometry which is easily manufactured. The cell (10) is made up of a plurality of body members (12), (14), (16), (18) which are bonded, e.g. fused, together such that a flow through passage is formed between facing surfaces of the body members via grooves (20), (28), (22). One of the body members (18) is shaped such that it acts as a lens for focussing excitation light into the passage (28) and another of the body members (16) is transparent, or has a transparent portion, for transmission of luminescence light out of passage (28).

Description

FLOW THROUGH CELL FOR SPECTROFLUOROMETRY

Technical Field

The present invention relates to a flow through cell for use in a spectrofluorometer for analysis of dissolved substances in a flowing liquid stream. The invention is particularly useful with high performance liquid chromatography.

Background Flow through cells are used to detect the presence of various dissolved chemical substances that are present in varying concentrations in a flowing liquid stream such as, for example, the effluent of a high-performance liquid chromatograph. If such chemical substances are luminescent when exposed to appropriate excitation radiation, they can be detected by spectrofluorometry. To obtain good resolution of different chemical substances eluted from the column of a chromatograph it is important that the effluent stream should undergo as little mixing as possible in its passage through the flow through cell. This is favoured by keeping the volume of the cell small and by ensuring that all parts of the conduit or passage through the cell are efficiently swept by the flowing liquid. Furthermore, for sensitive detection of a dissolved chemical substance by spectrofluorometry, it is desirable that the contents of the flow through cell be irradiated with the full intensity of the excitation light beam of the spectrofluorometer. Ideally, the size and shape of the cell should be matched to that of the excitation beam. This requirement is in conflict with the previously stated requirement of keeping the volume of the cell small.

In known flow cells it is usual to sacrifice efficiency of illumination so that the internal volume can be kept small. However, it is also known from United States Patent No. 5,493,405 by Keith Hulme to use a lens in a small volume flow through cell for concentrating the excitation light beam that is incident thereon into the sample chamber. The flow through cell of this patent, however, would be relatively difficult to manufacture because it involves the drilling of several holes through a block of black quartz to provide a sampling chamber having inlet and outlet ports. Additionally, these drilled holes have to be accurately located relative to each other such that they appropriately intersect for a flow through passage to be formed that does not have any imperfections such as may cause an undesirable amount of mixing in a fluid stream as it passes through the cell.

The discussion herein of the background to the invention is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was known or part of the common general knowledge in Australia as at the priority date of any of the claims.

Disclosure of the Invention

An object of the present invention is to provide a relatively easily manufacturable flow through cell for spectrofluorometry having a small volume flow through passage and which incorporates a lens for focussing an excitation beam of light into the passage.

Accordingly, the invention provides a flow through cell for use in a spectrofluorometer for analysis of dissolved chemical substances in a flowing liquid stream, including a body having a small volume flow through passage, wherein the body comprises a plurality of body members bonded together such that the flow through passage is formed between facing surfaces of at least two of said plurality of body members via a groove formed in a surface of at least one of the at least two body members, wherein one of said plurality of body members is shaped such that it acts as a lens for focussing a beam of excitation light into the small volume flow through passage, and wherein another of said plurality of body members, or a portion thereof, is transparent for transmission of luminescence light from chemical substances in the flowing liquid stream for detection.

Formation of the flow through cell from a plurality of body members, which may be formed for example from an optical grade quartz, offers several advantages in that appropriately shaped body members can be readily provided and the cell is readily formable, which saves costs. In particular, forming a flow through passage between facing surfaces of the body members via a groove formed in a surface of at least one of the facing body members allows for relatively easier manufacture of the flow through cell compared to the drilling of several holes through a block of quartz as in the above described prior U.S. patent. It also allows avoidance of adhesives in bonding the members together, that is, preferably the body members are bonded by heat and pressure to form the body. Use of adhesives is generally sought to be avoided because they may possibly fluoresce and thus contaminate the spectrofluorometric measurements. It is to be understood, however, that the invention does not exclude the possible use of adhesives to bond the members together, provided suitable non-fluorescing adhesives are used.

The flow through passage of the cell should be shaped to ensure that it is wholly swept by the flowing liquid stream to thereby avoid the formation of "pockets" of relatively slower moving or still liquid. The volume of the flow through cell should be small (for example in the order of 10-20 microlitres) commensurate with adequate sensitivity and signal to noise ratio. Thus the invention allows for sensitive detection of luminescent substances in a flowing liquid stream while preserving' the resolution of different substances present in consecutive parts of the flowing stream.

Preferably the flow through passage extends longitudinally and the body member which is shaped such that it acts as a lens is one of said at least two body members between which the flow through passage is formed. More preferably the body member which is shaped such that it acts as a lens includes the groove formed in a surface thereof. These features further simplify the manufacturability of the flow through cell in that the body member which is shaped such that it acts as a lens may be of a shape, such as generally semi- annular in cross section, that is more easily provided than a generally spherical shaped lens as in the prior art. Furthermore, this arrangement enables an optimum configuration for fluorescence measurements in that the body member, or portion thereof, which is transparent for transmission of luminescence (that is, fluorescence) light can be located relative to the body member which acts as a lens for the luminescence light to be detected orthogonally of the excitation light, as is disclosed in the detailed description hereinbelow.

Further features and advantages of the invention will become apparent from the following description with reference to the accompanying drawings of a preferred embodiment thereof, which is given by way of non-limiting example only for a better understanding of the invention and to show how it may be carried into effect.

Brief Description of the Drawings

Fig. 1 is an exploded perspective view of a plurality of body members for forming the flow cell embodiment and

Fig. 2 is an exploded perspective view of the assembled flow cell of Fig. 1 with a housing for mounting the flow cell and inlet and outlet connectors.

Detailed Description

The Illustrated flow through cell 10 is constructed from four body members 12, 14, 16, 18 which when bonded together form a body. Body member 12 is of rectangular parallelepiped shape and includes two semi- cylindrical recesses 20, 22 formed in opposite side faces 24, 26 thereof. Body members 14 and 16 are flat plates which are fused to the side faces 24, 26 of body member 12 for the recesses 20, 22 to thereby form an inlet port and outlet port for the flow through cell 10. The fourth body member 18 is constructed of optical grade quartz or other suitable material and has a semi-annular cross- sectional shape whereby a semi-cylindrical recess 28 is defined which extends the length of body member 18. When body member 18 is bonded to face 30 of body member 12 a flow through passage is formed which extends between the inlet and outlet ports 20, 22. End faces 32 of body member 18 are bonded to side body members 14 and 16 to close off each end of the flow through passage 28. Body member 18 is transparent and is so shaped such that an excitation beam of light falling on its outer curved surface is at least partially focussed into the flow through passage 28 of the body formed by the assembly of the four body members 12-18. Thus the body member 18 is shaped such that it acts as a lens.

Body member 16 is transparent and formed of optical grade quartz or other suitable material to provide a window for transmission of luminescence light from chemical substances dissolved within a liquid flowing through the cell for detection by the spectrofluorometer. Alternatively, transparent body member 16 may be so shaped as to focus light emitted from a region directly behind it into the detection optics of the spectrofluorometer. The faces of body members 12 and 14 that face into the flow through passage 28 may be provided with a reflective coating so that light is reflected back into the passage 28 rather than being lost through body members 12 and 14 if they be constructed of transparent material, or absorbed by body members 12 and 14 if they be constructed of opaque material. Preferably body members 12 and 14 are constructed of transparent material and have external faces parallel to the faces forming part of the passage 28. The reflective coating can be applied to the external face, thereby being protected from contact with material passing through the passage 28. Such protection is desirable to protect the reflective surface from corrosion by the material in the passage 28 and to protect the material in the passage from contamination by the reflective surface.

The body members 12, 14, 16, 18 are sealingly bonded together by application of heat and pressure, that is, they are fused together.

The flow through cell 10 formed by the fused assembly of body members

12-18 is mounted in a housing 34 constructed of aluminium or other suitable material. The housing 34 includes a channel 36 which is shaped to receive the flow through cell 10 and provide inwardly facing surfaces 38 against which top and bottom portions of surface 30 of body member 12 (see references 40 on Fig. 2) bear. Cell 10 is held in housing 34 by a securing screw 42 which passes through threaded hole 44 in housing 34 to push body member 12 against faces 38. Inlet and outlet connectors 46 and ferrules 48 are installed in holes 50 of housing 34 in such a way as to make sealing contact with, respectively, the inlet and outlet ports 20 and 22 of flow through cell 10.

Housing 34 is so constructed that when it together with housed flow through cell 10 is properly placed in the sample compartment of an appropriate spectrofluorometer, body member 18 faces the excitation beam of the spectrofluorometer and body member 16 faces the optical elements of the spectrofluorometer that accept emitted light. Thus the cell 10 configuration is optimised for taking fluorescence measurements. Inlet and outlet connectors 46 are respectively connected to the source of the liquid stream, which may be on the effluent tube of a liquid chromatograph or other source of liquid for analysis, and to means for receiving the liquid stream after its passage through the cell 10.

The flow through passage 28 is of small volume, for example less than or about 20 microlitres. An example size for body member 18 which acts as a lens is 10mm long, 1 mm internal radius and 3 mm external radius, which gives a volume for flow through passage 28 of about 15.7 microlitres. The inlet and outlet ports 20, 22 are generally correspondingly sized and may have volumes of only about 1.5 microlitres each, for example they may have a groove radius of 0.5 mm and be about 4 mm length. The cylindrical lens shaped surface of body member 18 increases the excitation light flux density in the sample in passage 28. The fluorescence is viewed along the long dimension of passage 28. Tests indicate a factor of two (2) improvement in cell 10 sensitivity compared to a cell with flat walls.

It will be appreciated that the invention as above described admits of very simple manufacture, for example, the recesses 20, 22 and 28, being semi- cylindrical, may be readily formed in the surfaces of the body members 12 and 18. The surfaces of the flow through passage, being exposed prior to fusing the. body members, are also easier to polish (as may be required) than would be the flow passage surfaces of a cell as in the above described prior US patent. A flow through passage as described is also efficiently swept by the liquid flowing therethrough. It is to be understood that other than semicylindrical shapes for the ports 20, 22 and passage 28 may be used, for example 'V shaped cross- sectional recesses are satisfactory. Furthermore, the transparent portion or window for transmission of luminescence light from the passage 28 may be via the body member 14 instead of body member 16, or via another portion of the body comprised of members 12-18. Also the dimensions given for body member 18 hereinbefore are merely one example size determined in development of the flow through cell.

The invention described herein is susceptible to variations, modifications and/or additions other than those specifically described and it is to be understood that the invention includes all such variations, modifications and/or additions which fall within the scope of the following claims.

Claims

1. A flow through cell for use in a spectrofluorometer for analysis of dissolved chemical substances in a flowing liquid stream, including a body having a small volume flow through passage, wherein the body comprises a plurality of body members bonded together such that the flow through passage is formed between facing surfaces of at least two of said plurality of body members via a groove formed in a surface of at least one of the at least two body members, wherein one of said plurality of body members is shaped such that it acts as a lens for focussing a beam of excitation light into the small volume flow through passage, and wherein another of said plurality of body members, or a portion thereof, is transparent for transmission of luminescence light from chemical substances in the flowing liquid stream for detection.

2. A flow through cell according to claim 1 wherein the flow through passage extends longitudinally and the body member which is shaped such that it acts as a lens is one of said at least two body members between which the flow through passage is formed.

3. A flow through cell according to claim 2 wherein the body member which is shaped such that it acts as a lens includes the groove formed in a surface thereof.

4. A flow through cell according to claim 3 wherein the body member which is shaped such that it acts as a lens and includes the groove is generally semi- annular in cross-sectional shape.

5. A flow through cell according to claim 4 wherein said another body member, or said portion thereof, which is transparent for transmission of luminescence light is located relative to the body member which is shaped such that it acts as a lens for the luminescence light to be detected orthogonally of the beam of excitation light.

6. A flow through cell according to any one of claims 1 to 5 wherein the flow through passage includes an outlet port substantially at a right-angle to the flow through passage into which excitation light is focussed, the outlet port also being formed between facing surfaces of two of said plurality of body members via a grove formed in a surface of at least one of those two body members, whereby there are at least three body members.

7. A flow through cell according to claim 6 wherein one of the body members which forms the outlet port is orthogonal to the body member which is shaped such that it acts as a lens and is the body member which is transparent or which includes a transparent portion, whereby luminescence light is detectable orthogonally of the beam of excitation light.

8. A flow through cell according to claim 6 or 7 wherein the flow through passage includes an inlet port substantially at a right-angle to the flow through passage into which excitation light is focussed, the inlet port extending substantially parallel to the outlet port and also being formed between facing surfaces of two of said plurality of body members via a groove formed in a surface of at least one of those two body members, whereby there are four body members.

9. A flow through cell according to claim 8 wherein one body member is of rectangular parallelepiped shape and includes a groove in opposite surfaces for forming said inlet and outlet ports, wherein the body member which is shaped such that it acts as a lens includes a longitudinal groove for forming said flow through passage into which excitation light is focussed, and is bonded to a flat surface of said one body member of rectangular parallelepiped shape, and the other two body members are flat plates bonded to said opposite surfaces of the body member of rectangular parallelepiped shape and to end faces of the body member which is shaped such that it acts as a lens such that a flow through passage is formed via the grooves.

10. A flow through cell according to claims 1 to 9 wherein the groove or the grooves are semi-cylindrical.

11. A flow through cell according to claims 1 to 10 wherein the plurality of body members are bonded together by fusing using heat and pressure.