JP4661768B2 - Cell device for fluorescence measurement and fluorescence detector - Google Patents

Cell device for fluorescence measurement and fluorescence detector Download PDF

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JP4661768B2
JP4661768B2 JP2006302519A JP2006302519A JP4661768B2 JP 4661768 B2 JP4661768 B2 JP 4661768B2 JP 2006302519 A JP2006302519 A JP 2006302519A JP 2006302519 A JP2006302519 A JP 2006302519A JP 4661768 B2 JP4661768 B2 JP 4661768B2
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昌秀 軍司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0378—Shapes
    • G01N2021/0382—Frustoconical, tapered cell
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6482—Sample cells, cuvettes

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Description

本発明は、蛍光検出に用いられるキュベットや高速液体クロマトグラフ用の蛍光検出器に用いられるフローセルに関するものである。   The present invention relates to a flow cell used in a cuvette used for fluorescence detection or a fluorescence detector for high performance liquid chromatography.

蛍光検出の原理は、セル中の試料に励起光を照射することで試料から蛍光を発生させ、この蛍光を検出器により検出するものである。放射される蛍光量は、セル中の試料に入射する励起光量が多いほど多くなる。
フローセルについて説明すると、試料に励起光を入射させる従来技術としては、図３（Ａ）に示すようにフローセルの側面から励起光を液体試料に入射させる方法と、図３（Ｂ）に示すようにフローセルの長手方向から励起光を入射させる方法がある(特許公報１参照。)。どちらの方法もフローセルの一面側から励起光を入射させるのみであるため、効率が悪い。 The principle of fluorescence detection is that a sample in a cell is irradiated with excitation light to generate fluorescence from the sample, and this fluorescence is detected by a detector. The amount of fluorescence emitted increases as the amount of excitation light incident on the sample in the cell increases.
The flow cell will be described. As a conventional technique for making the excitation light incident on the sample, as shown in FIG. 3A, the excitation light is made incident on the liquid sample from the side surface of the flow cell, and as shown in FIG. There is a method in which excitation light is incident from the longitudinal direction of the flow cell (see Patent Publication 1). Both methods are inefficient because they only allow excitation light to enter from one side of the flow cell.

また、基端と末端とを有するフローセルにおいて、基端に励起レンズを備え、末端に逆反射レンズを備えることで、基端側から照射された励起光を集光し、励起光の光路長を２倍にする蛍光検出器も提案されている（特許文献２参照。）。
特許第３８４４７３８号公報 特表２００４−５３０８６８号公報 In addition, in a flow cell having a base end and a terminal end, an excitation lens is provided at the base end and a retroreflective lens is provided at the end, so that the excitation light irradiated from the base end side is collected and the optical path length of the excitation light is reduced. A fluorescence detector that doubles has also been proposed (see Patent Document 2).
Japanese Patent No. 3844738 JP-T-2004-530868

蛍光検出器の感度を向上させるためには、セル中の試料に対して出来るだけ多くの励起光を効率的に入射させる必要があり、また発生した蛍光についても出来るだけ効率的に検出器へ集光させる必要がある。しかし、上述の特許文献２は、セルの基端側からの励起光のみを集光するものである。   In order to improve the sensitivity of the fluorescence detector, it is necessary to efficiently make as much excitation light as possible incident on the sample in the cell, and the generated fluorescence is collected to the detector as efficiently as possible. It is necessary to light up. However, the above-mentioned Patent Document 2 collects only excitation light from the base end side of the cell.

そこで本発明は、照射された励起光と発生した蛍光をできるだけ効率的に検出器へ集光させ、光路長を長くすることができる蛍光測定用セル装置を提供することを目的とする。   Accordingly, an object of the present invention is to provide a fluorescence measuring cell device capable of condensing irradiated excitation light and generated fluorescence to a detector as efficiently as possible to increase the optical path length.

本発明の蛍光測定用セル装置は、試料水が入れられ又は流通する角型のセル本体を備え、セル本体の１つの側面から励起光が照射されるセルと、上記セルの４側面の内の励起光入射側に対向する側面側に配置され、セルを透過した励起光をセル本体方向に反射させる第１ミラーと、励起光入射側の側面に隣接する１つの側面に対向して配置され、セル本体内の試料水から発せられた蛍光をセル本体方向に反射させる第２ミラーと、セル本体の１つの底面側にその底面と励起光入射方向とにともに向けて傾斜して配置され、セル本体に照射された励起光の内、セル本体から外れた励起光をセル本体の底面から入射させるように反射させる第３ミラーと、を備えている。   The cell device for fluorescence measurement of the present invention includes a rectangular cell body into which sample water is put or circulated, a cell irradiated with excitation light from one side surface of the cell body, A first mirror disposed on the side facing the excitation light incident side, reflecting the excitation light transmitted through the cell in the direction of the cell body, and disposed on one side adjacent to the side on the excitation light incident side; A second mirror that reflects the fluorescence emitted from the sample water in the cell body in the direction of the cell body, and one bottom surface side of the cell body that is inclined toward both the bottom surface and the excitation light incident direction; And a third mirror that reflects the excitation light that is off the cell main body and is incident on the bottom surface of the cell main body.

励起光又は蛍光を反射させることで検出効率を高めるため、第３ミラーが配置されている底面とは反対側の底面に対向して第４ミラーを配置するようにしてもよい。第４ミラーは第３ミラーにより反射されて上記セル本体内を透過してきた励起光を上記セル本体方向に反射させるものである。   In order to enhance detection efficiency by reflecting excitation light or fluorescence, the fourth mirror may be disposed to face the bottom surface opposite to the bottom surface on which the third mirror is disposed. The fourth mirror reflects the excitation light reflected by the third mirror and transmitted through the cell body toward the cell body.

励起光を効率よくセル本体に照射し、又は発光を効率よく受光光学系で受光できるようにするため、第１ミラー、第２ミラー、第３ミラー及び第４ミラーの内の少なくとも１つを、セル本体内に光を集光させる凹面ミラーにしてもよい。   In order to efficiently irradiate the cell body with the excitation light or to receive the light emission efficiently with the light receiving optical system, at least one of the first mirror, the second mirror, the third mirror, and the fourth mirror is provided. A concave mirror that collects light in the cell body may be used.

本発明のセルはキュベットと称される有底のセルと、フローセルの両方を含む。フローセルは試料導入口及び試料排出口をもつ。   The cell of the present invention includes both a bottomed cell called a cuvette and a flow cell. The flow cell has a sample inlet and a sample outlet.

本発明の蛍光検出器は、蛍光測定用セル装置と、セル本体に励起光を入射させる励起光学系と、第２ミラーが配置されている側の側面とは反対側の側面側に配置され、セル本体内の試料水から発生した蛍光を受光する受光光学系と、を備えている。   The fluorescence detector of the present invention is disposed on the side of the side opposite to the side on which the second mirror is disposed, the cell device for fluorescence measurement, the excitation optical system that makes the excitation light incident on the cell body, And a light receiving optical system for receiving fluorescence generated from the sample water in the cell main body.

本発明は、セルと、セルの側面に設けられた第１ミラー、第２ミラー及び第３ミラーとを備えたので、光路長が長くなり、試料水に照射される励起光と試料水から発せられる発光の量が多くなるので、これまでよりも効率的に受光側に光を集光することが可能となる。   Since the present invention includes the cell and the first mirror, the second mirror, and the third mirror provided on the side surface of the cell, the optical path length is increased, and the excitation light and the sample water emitted from the sample water are emitted. Since the amount of emitted light is increased, it is possible to collect light on the light receiving side more efficiently than before.

第４ミラーを備えるようにすれば、より多くの蛍光を受光側に集光することができるようになる。   If the fourth mirror is provided, more fluorescence can be collected on the light receiving side.

ミラーの内の少なくとも１つを凹面ミラーにすると、セル本体内に光を集光しやすくなり、検出効率を向上させることができる。   If at least one of the mirrors is a concave mirror, light can be easily collected in the cell body, and detection efficiency can be improved.

フローセルとして用いることにより、試料水を連続的に供給することができるようになる。   By using as a flow cell, sample water can be continuously supplied.

本発明の蛍光測定用セル装置と、励起光学系と、受光光学系とを備えるようにすれば、これまでよりも効率的に蛍光を検出することができるようになる。   If the fluorescence measuring cell device of the present invention, the excitation optical system, and the light receiving optical system are provided, the fluorescence can be detected more efficiently than before.

以下に本発明の実施例を詳細に説明する。
図１は本発明の蛍光測定用セル装置を示しており、（Ａ）は上面図、（Ｂ）は斜視図、（Ｃ）は側面図である。
フローセル２１は上下方向に長く、下方向の面は水平面に対して角度をなして配置されている直方体である。セル２１の側面には第１ミラー１及び第２ミラー２が隣接して備えられている。また、セル２１の下面には第３ミラー３、上面には第４ミラー４が備えられている。
ミラー１は４側面の内の励起光入射側に対向する側面に配置され、ミラー２はミラー１及び励起光入射側の側面に隣接して配置されている。 Examples of the present invention will be described in detail below.
FIG. 1 shows a cell device for fluorescence measurement according to the present invention, wherein (A) is a top view, (B) is a perspective view, and (C) is a side view.
The flow cell 21 is a rectangular parallelepiped that is long in the vertical direction and whose lower surface is arranged at an angle with respect to the horizontal plane. A first mirror 1 and a second mirror 2 are provided adjacent to the side surface of the cell 21. A third mirror 3 is provided on the lower surface of the cell 21 and a fourth mirror 4 is provided on the upper surface.
The mirror 1 is disposed on the side surface facing the excitation light incident side among the four side surfaces, and the mirror 2 is disposed adjacent to the mirror 1 and the side surface on the excitation light incident side.

セル２１内には試料水が入れられ又は流通するセル本体２２が設けられており、セル本体２２の一端は試料導入口２３、他端は試料排出口２４となっている。試料導入口２３及び試料排出口２４は、フローセル２１のどの面に設けてもよい。
ミラー３は、セル２１の側面から照射された励起光１３ａをセル本体２２と平行方向に反射させるために、セル２１の底面と励起光入射方向に対して約４５°の角度をなして配置されている。これにより励起光をミラー３でセル本体２２方向に反射させることができる。 A cell main body 22 into which sample water is put or circulated is provided in the cell 21, and one end of the cell main body 22 is a sample introduction port 23 and the other end is a sample discharge port 24. The sample introduction port 23 and the sample discharge port 24 may be provided on any surface of the flow cell 21.
The mirror 3 is disposed at an angle of about 45 ° with respect to the bottom surface of the cell 21 and the excitation light incident direction in order to reflect the excitation light 13 a irradiated from the side surface of the cell 21 in a direction parallel to the cell body 22. ing. Thereby, the excitation light can be reflected by the mirror 3 toward the cell body 22.

ミラー１，２，３，４は、例えば蒸着により形成することができる。また、ミラー１，２，３，４はセル本体２２方向に光を集光させる凹面ミラーとしてもよい。
上述のセルとしてはフローセルを示したが、試料導入口２３及び試料排出口２４が兼用される角型セルを用いることもできる。 The mirrors 1, 2, 3, and 4 can be formed by vapor deposition, for example. The mirrors 1, 2, 3, and 4 may be concave mirrors that collect light in the direction of the cell body 22.
Although a flow cell is shown as the above-described cell, a square cell in which the sample introduction port 23 and the sample discharge port 24 are also used can be used.

次に同実施例の動作を説明する。セル２１の側面の光源側から励起光を入射させると、入射した一部の励起光１１ａは、セル２１内のセル本体２２を通過した後にミラー１によって反射され、反射された励起光１１ｂは、再びセル本体２２内を通過する。これにより発光１５が生じる。
また一部の励起光１３ａはセル本体２２に直接照射されないが、セル２１の側面を通過し、ミラー３によってセル２１の軸と平行な方向に反射し、反射された励起光１３ｂはセル本体２２内の試料に照射され、発光１５を生じる。 Next, the operation of this embodiment will be described. When excitation light is incident from the light source side of the side surface of the cell 21, the part of the incident excitation light 11a is reflected by the mirror 1 after passing through the cell body 22 in the cell 21, and the reflected excitation light 11b is It passes through the cell body 22 again. As a result, light emission 15 occurs.
A part of the excitation light 13a is not directly irradiated to the cell body 22, but passes through the side surface of the cell 21 and is reflected by the mirror 3 in a direction parallel to the axis of the cell 21, and the reflected excitation light 13b is reflected in the cell body 22. The sample inside is irradiated to generate luminescence 15.

一部の励起光１３ｂは、セル２１の長手方向からセル本体２２を通過し、さらにミラー４によって反射される。ミラー４で反射された励起光１３ｃは、セル本体２２内の試料に照射され、発光１５を生じる。
生じた蛍光１５の一部は直接検出器側へ出射し、また一部の発光１２ａはミラー２で反射された後に検出器側へと出射される。
尚、本実施例で使用されるミラーは、光を効率的に収束させるため、曲面形状にしてもよい。 A part of the excitation light 13 b passes through the cell body 22 from the longitudinal direction of the cell 21 and is reflected by the mirror 4. The excitation light 13 c reflected by the mirror 4 is irradiated on the sample in the cell main body 22 to generate light emission 15.
A part of the generated fluorescence 15 is directly emitted to the detector side, and a part of the emitted light 12a is reflected by the mirror 2 and then emitted to the detector side.
Note that the mirror used in this embodiment may have a curved surface shape in order to converge light efficiently.

次に本発明の蛍光検出器についての実施例を図２を参照しながら説明する。
セル２１の励起光入射側には白色光の光源３１が配置され、光学レンズ３２と、白色光から目的の波長の励起光として分光する回折格子３３と、励起光を集光する光学レンズ３４とを介して、励起光がセル２１内に入射されるようになっている。 Next, an embodiment of the fluorescence detector of the present invention will be described with reference to FIG.
A white light source 31 is disposed on the excitation light incident side of the cell 21, an optical lens 32, a diffraction grating 33 that separates white light as excitation light having a target wavelength, and an optical lens 34 that collects the excitation light. The excitation light is incident on the cell 21 via the.

光学レンズ３２は光源３１の白色光を平行光に集光する球面レンズ、光学レンズ３４は励起光をセル２１内のセル本体に集光できるシリンダー状レンズであることが好ましい。
セル２１の蛍光出射側には光検出器３５が配置され、発光を集光する光学レンズ３６と、発光を回折する回折格子３７と、回折光を集光する光学レンズ３８を介して、光検出器３５に導かれるようになっている。 The optical lens 32 is preferably a spherical lens that condenses the white light of the light source 31 into parallel light, and the optical lens 34 is preferably a cylindrical lens that can condense the excitation light on the cell body in the cell 21.
A light detector 35 is disposed on the fluorescence emission side of the cell 21 and detects light through an optical lens 36 that condenses the emitted light, a diffraction grating 37 that diffracts the emitted light, and an optical lens 38 that condenses the diffracted light. It is guided to the container 35.

次に同実施例の動作を説明する。
光源３１から発生した白色光から回折格子３３によって目的の波長の光を励起光として分光する。分光された励起光はレンズ３４によって集光されフローセル２１へ入射する。
入射した励起光は図１の反射ミラー１〜４によって反射され、効率的にセル本体内へ入射する。励起光によって発生した蛍光の一部は直接セル２１から蛍光用回折格子３７へと出射し、また一部は反射ミラーによって反射された後に回折格子３７へと出射される。出射された蛍光は回折格子３７によって分光され、目的の波長の光のみが光検出器３５へと集光される。 Next, the operation of this embodiment will be described.
From the white light generated from the light source 31, the diffraction grating 33 separates light having a target wavelength as excitation light. The split excitation light is collected by the lens 34 and enters the flow cell 21.
The incident excitation light is reflected by the reflection mirrors 1 to 4 in FIG. 1 and efficiently enters the cell body. Part of the fluorescence generated by the excitation light is directly emitted from the cell 21 to the fluorescence diffraction grating 37, and part of the fluorescence is reflected by the reflection mirror and then emitted to the diffraction grating 37. The emitted fluorescence is spectrally separated by the diffraction grating 37 and only light having a target wavelength is collected on the photodetector 35.

本発明は、フローセル側面から入射した励起光をフローセル長軸方向へと反射させるミラー面を設置し、また、フローセルの側面およびもう一方の端面にミラーを設置することで、セル本体を通過した励起光を反射し、再びフローセル内に入射させるものである。フローセル側面にミラー面を配置し、試料から発生した蛍光の一部を反射することで、効率的に蛍光を光検出器へと集光させることができる。   The present invention provides a mirror surface that reflects excitation light incident from the side surface of the flow cell in the direction of the long axis of the flow cell, and a mirror is provided on the side surface and the other end surface of the flow cell, so that the excitation that has passed through the cell body It reflects light and makes it enter the flow cell again. By arranging a mirror surface on the side surface of the flow cell and reflecting a part of the fluorescence generated from the sample, the fluorescence can be efficiently collected on the photodetector.

本発明は、フローセル内の試料に対し、フローセル側面及び長軸方向から励起光を入射させ、さらにミラーによりセルを通過した光を再び試料に入射させているので、試料に入射する励起光量は前述の２つの従来技術を足し合わせた量になる。また、試料から発生した蛍光の一部も反射鏡によって検出器へと反射され、従来技術よりも多くの蛍光が検出器へと導かれる。その結果、従来技術に比べ蛍光検出器としての装置感度が向上する。   In the present invention, the excitation light is incident on the sample in the flow cell from the side surface and the long axis direction of the flow cell, and the light passing through the cell is again incident on the sample by the mirror. This is the sum of these two conventional technologies. In addition, a part of the fluorescence generated from the sample is also reflected by the reflecting mirror to the detector, and more fluorescence is guided to the detector than in the prior art. As a result, the sensitivity of the apparatus as a fluorescence detector is improved as compared with the prior art.

ミラーの数を多くし、光を反射させるようにしたので、試料に入射する励起光の量と、検出器へと出射される蛍光の量が増加し、従来技術に比べてより高感度な蛍光検出器が実現できる。   Since the number of mirrors is increased and light is reflected, the amount of excitation light incident on the sample and the amount of fluorescence emitted to the detector increase, and fluorescence with higher sensitivity compared to the conventional technology. A detector can be realized.

本発明は、蛍光検出に用いられるキュベットや高速液体クロマトグラフ用の蛍光検出器に用いられるフローセルに利用することができる。   The present invention can be used for a cuvette used for fluorescence detection and a flow cell used for a fluorescence detector for high performance liquid chromatography.

本発明の蛍光測定用セル装置を示しており、（Ａ）は上面図、（Ｂ）は斜視図、（Ｃ）は側面図である。1 shows a fluorescence measurement cell device according to the present invention, in which (A) is a top view, (B) is a perspective view, and (C) is a side view. 本発明の蛍光検出器についての概略構成図である。It is a schematic block diagram about the fluorescence detector of this invention. （Ａ）及び（Ｂ）は、従来用いられているフローセルの概略構成図である。(A) And (B) is a schematic block diagram of the flow cell used conventionally.

符号の説明Explanation of symbols

１〜４ ミラー
１１ａ，１１ｂ，１３ａ，１３ｂ，１３ｃ 励起光
１２ａ，１５ 発光
２１ セル
２２ セル本体
２３ 試料導入口
２４ 試料排出口 1-4 Mirror 11a, 11b, 13a, 13b, 13c Excitation light 12a, 15 Light emission 21 Cell 22 Cell body 23 Sample inlet 24 Sample outlet

Claims (5)

試料水が入れられ又は流通する角型のセル本体を備え、前記セル本体の１つの側面から励起光が照射されるセルと、
前記セルの４側面の内の励起光入射側に対向する側面側に配置され、前記セルを透過した励起光を前記セル本体方向に反射させる第１ミラーと、
前記励起光入射側の側面に隣接する１つの側面に対向して配置され、前記セル本体内の試料水から発せられた蛍光を前記セル本体方向に反射させる第２ミラーと、
前記セル本体の１つの底面側にその底面と励起光入射方向とにともに向けて傾斜して配置され、前記セル本体に照射された励起光の内、前記セル本体から外れた励起光を前記セル本体の底面から入射させるように反射させる第３ミラーと、
を備えた蛍光測定用セル装置。 A square cell body into which sample water is put or circulated, and a cell irradiated with excitation light from one side surface of the cell body;
A first mirror that is disposed on a side surface facing the excitation light incident side of the four side surfaces of the cell and reflects the excitation light transmitted through the cell toward the cell body;
A second mirror that is disposed to face one side surface adjacent to the side surface on the excitation light incident side and reflects fluorescence emitted from the sample water in the cell body toward the cell body;
The cell body is disposed on one bottom surface so as to be inclined toward both the bottom surface and the excitation light incident direction, and among the excitation light irradiated to the cell body, the excitation light that is off the cell body is emitted from the cell body. A third mirror that reflects so as to be incident from the bottom surface of the main body;
A cell device for fluorescence measurement comprising: 第３ミラーが配置されている底面とは反対側の底面に対向して配置され、第３ミラーにより反射されて前記セル本体内を透過してきた励起光を前記セル本体方向に反射させる第４ミラーをさらに備えた請求項1に記載の蛍光測定用セル装置。   A fourth mirror that is disposed to face the bottom surface opposite to the bottom surface on which the third mirror is disposed, and that reflects the excitation light reflected by the third mirror and transmitted through the cell body toward the cell body. 2. The fluorescence measuring cell device according to claim 1, further comprising: 第１ミラー、第２ミラー、第３ミラー及び第４ミラーの内の少なくとも１つは、前記セル本体内に光を集光させる凹面ミラーである請求項１又は２に記載の蛍光測定用セル装置。   3. The fluorescence measuring cell device according to claim 1, wherein at least one of the first mirror, the second mirror, the third mirror, and the fourth mirror is a concave mirror that collects light in the cell body. 4. . 前記セルは試料導入口及び試料排出口が形成されたフローセルである請求項１から３のいずれか一項に記載の蛍光測定用セル装置。   The fluorescence measurement cell device according to any one of claims 1 to 3, wherein the cell is a flow cell in which a sample introduction port and a sample discharge port are formed. 請求項１から４のいずれか一項に記載の蛍光測定用セル装置と、
前記セル本体に励起光を入射させる励起光学系と、
第２ミラーが配置されている側の側面とは反対側の側面側に配置され、前記セル本体内の試料水から発生した蛍光を受光する受光光学系と、
を備えた蛍光検出器。 A cell device for fluorescence measurement according to any one of claims 1 to 4,
An excitation optical system for causing excitation light to enter the cell body;
A light receiving optical system disposed on the side surface opposite to the side surface on which the second mirror is disposed, and receiving fluorescence generated from the sample water in the cell body;
Fluorescence detector with
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