JPH0342550A - Fluorescent detector for high-performance liquid chromatography - Google Patents
Fluorescent detector for high-performance liquid chromatographyInfo
- Publication number
- JPH0342550A JPH0342550A JP17720489A JP17720489A JPH0342550A JP H0342550 A JPH0342550 A JP H0342550A JP 17720489 A JP17720489 A JP 17720489A JP 17720489 A JP17720489 A JP 17720489A JP H0342550 A JPH0342550 A JP H0342550A
- Authority
- JP
- Japan
- Prior art keywords
- laser
- titanium
- sapphire
- light
- nonlinear optical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004128 high performance liquid chromatography Methods 0.000 title claims description 6
- 229910052594 sapphire Inorganic materials 0.000 claims abstract description 16
- 239000010980 sapphire Substances 0.000 claims abstract description 16
- 239000013078 crystal Substances 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 10
- 230000003287 optical effect Effects 0.000 claims abstract description 9
- 239000010936 titanium Substances 0.000 abstract description 18
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 abstract description 17
- 229910052719 titanium Inorganic materials 0.000 abstract description 17
- 239000000975 dye Substances 0.000 abstract description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 abstract description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052802 copper Inorganic materials 0.000 abstract description 4
- 239000010949 copper Substances 0.000 abstract description 4
- 239000004065 semiconductor Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000000862 absorption spectrum Methods 0.000 abstract description 3
- 239000007850 fluorescent dye Substances 0.000 abstract description 3
- 238000001215 fluorescent labelling Methods 0.000 abstract description 3
- 229910052786 argon Inorganic materials 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract 2
- 230000010287 polarization Effects 0.000 abstract 1
- 230000005284 excitation Effects 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 2
- 230000002999 depolarising effect Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- XJCVRTZCHMZPBD-UHFFFAOYSA-N 3-nitroaniline Chemical compound NC1=CC=CC([N+]([O-])=O)=C1 XJCVRTZCHMZPBD-UHFFFAOYSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- GQYHUHYESMUTHG-UHFFFAOYSA-N lithium niobate Chemical compound [Li+].[O-][Nb](=O)=O GQYHUHYESMUTHG-UHFFFAOYSA-N 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000001782 photodegradation Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Lasers (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、高速液体クロマトグラフィー（以下ＨＰＬＣ
と略記する）用蛍光検出器に関する。更に詳しくは、レ
ーザー光の波長を連続的に変換可能なチタンサファイア
レーザーを励起光源に用いたＩＩＰＬＣ用蛍光検用益光
検出器。Detailed Description of the Invention [Industrial Field of Application] The present invention is directed to high performance liquid chromatography (hereinafter referred to as HPLC).
(abbreviated as )). More specifically, a useful light detector for fluorescence analysis for IIPLC uses a titanium sapphire laser as an excitation light source, which can continuously convert the wavelength of laser light.
［従来の技術］
＋１ＰＬＣ用蛍光検出器は、例えば医学、生化学の分野
に於いて、生理活性物質等の生体試料の高感度定量分析
、即ち検出限界をピコグラムからフェムトグラム又はピ
コモルからフェムトモルまで向上させることが考えられ
る検出器として広い応用が期待される機器である。[Prior Art] +1 Fluorescence detector for PLC is used for high-sensitivity quantitative analysis of biological samples such as physiologically active substances in the fields of medicine and biochemistry, that is, improving the detection limit from picogram to femtogram or from picomole to femtomole. This device is expected to have a wide range of applications as a detector that can be used to
１１ＰＬｃ用蛍光検出器の励起光源には、種々のレーザ
ーが用いられている。例えばガスレーザーとしてはＡ「
、ＩＩ ｃ −Ｎ ｅ銅蒸気レーザー、固体レーザーと
してはＹＡＧ、半導体レーザー、液体レーザーとしては
色素レーザー、更にＸｃクラン等々である。Various lasers are used as excitation light sources for 11PLc fluorescence detectors. For example, as a gas laser, A
, II c-N e copper vapor laser, solid lasers such as YAG and semiconductor lasers, liquid lasers such as dye lasers, and Xc clans.
上記、Ａ「、ｌ１ｅ−Ｎｅ、銅蒸気、ＹＡＧ等のレーザ
ーは波長を連続的に変換して用いることができない。The above-mentioned lasers such as A', l1e-Ne, copper vapor, YAG, etc. cannot be used by continuously converting the wavelength.
また、半導体レーザーでは数ｎｍ程度の範囲でしか変換
できず出力も低い。色素レーザーは可変域が５０〜１０
０ｔｖの幅であるが、色素の光劣化により、レーザー発
振出力の変動を起こし更に寿命が短く保守も容易でない
。Furthermore, semiconductor lasers can only convert within a range of several nanometers, and their output is low. Dye laser has a variable range of 50 to 10
Although the width is 0 tv, photodeterioration of the dye causes fluctuations in laser oscillation output, and furthermore, the lifespan is short and maintenance is not easy.
通常、試料の吸収スペクトルは上記のレーザーの波長と
異なっていることが多く、そのため、このようなレーザ
ーを装置に組み込んだ検出器を用いた場合、高感度の定
量分析（例えばピコグラムオーダー）を行うため蛍光ス
ペクトルを１Ｉ１１定する際に、試料を化学修飾するこ
とによって蛍光標識する必要がある。しかも、例え蛍光
標識したとしても、フェムトオーダーの高感度分析は困
難である。Normally, the absorption spectrum of a sample is often different from the wavelength of the laser mentioned above, so when using a detector that incorporates such a laser into the device, it is possible to perform highly sensitive quantitative analysis (e.g. picogram order). Therefore, when determining the fluorescence spectrum, it is necessary to fluorescently label the sample by chemically modifying it. Moreover, even if fluorescent labeling is used, high-sensitivity analysis on the femtoorder is difficult.
色素レーザーは可変地域が５０〜１００ｎａ＋の幅であ
るが、色素の光劣化によりレーザー発振出力の変動を起
こす。そのため、蛍光スペクトルを容易にかつ常時安定
に測定することが困難である。Although the dye laser has a variable range of 50 to 100 na+, the laser oscillation output varies due to photodegradation of the dye. Therefore, it is difficult to easily and always stably measure the fluorescence spectrum.
Ｘｅランプの光を分光して用いた場合、約２２０〜１０
００ｎ■の広い範囲の光が得られるが、光の強度がレー
ザー光に比較して弱くこれを用いて高感度定量分析する
ことは困難である。When using Xe lamp light, it is approximately 220 to 10
Although a wide range of light of 00 nm can be obtained, the intensity of the light is weaker than that of laser light, and it is difficult to perform high-sensitivity quantitative analysis using this light.
［問題を解決するための手段］
本発明者らは、上記した問題のないＩＩＰＬｃ用蛍光検
出器につき鋭意検討した結果、励起レーザー光の光源に
波長可変固体レーザーとして、レーザー媒質がチタンを
添加（異種元素としてドープ）したサファイアである、
いわゆるチタンサファイアレーザーを用いることにより
、前記問題点を解決できることを見い出だし本発明を完
成した。[Means for Solving the Problems] As a result of intensive study on a fluorescence detector for IIPLc that does not have the above-mentioned problems, the present inventors used a wavelength tunable solid-state laser as the light source of the excitation laser light, and the laser medium added titanium ( Sapphire doped with a foreign element,
The inventors have discovered that the above problems can be solved by using a so-called titanium sapphire laser, and have completed the present invention.
即ち本発明の目的は、被測定試料の蛍光スペクトルを選
択的に且つ安定した状態で容易に測定が可能なＨＰＬＣ
用蛍光検出器を堤供することである。That is, an object of the present invention is to provide an HPLC method that can easily measure the fluorescence spectrum of a sample to be measured selectively and in a stable state.
The purpose is to provide a fluorescence detector for
以下本発明を更に詳述する。The present invention will be explained in more detail below.
本発明では、被測定試料に照射するレーザー光の光源に
、波長がある範囲内で連続的に変えることのできる波長
可変固体レーザーを用いることを特徴とするが、ここで
用いる固体レーザーは、チタンを添加したサファイアで
あるチタンサファイアレーザーである。ここでサファイ
アへのチタンの添加量は、０．Ｏ１原子％〜０．５原子
％の範囲であることが望ましい。チタン添加量が０．Ｏ
１原子％より少ないと増幅度が小さくなりレーザー発振
が困難となる。また同添加量が０．５原子％より多くな
ると発光領域に存在する残留吸収が極端に大きくなり、
レーザー発振効率が低下する。また、発光寿命が短くな
り、そのためフラッシュランプでのチタンサファイアの
光ボンピングが困難となり、更にランプの使用寿命も短
くなる。The present invention is characterized in that a tunable solid-state laser whose wavelength can be changed continuously within a certain range is used as the light source of the laser light that irradiates the sample to be measured. This is a titanium sapphire laser, which is sapphire doped with. Here, the amount of titanium added to sapphire is 0. The O content is preferably in the range of 1 atomic % to 0.5 atomic %. The amount of titanium added is 0. O
When the amount is less than 1 atomic %, the amplification degree becomes small and laser oscillation becomes difficult. In addition, when the amount of addition exceeds 0.5 at%, the residual absorption existing in the light emitting region becomes extremely large.
Laser oscillation efficiency decreases. Also, the luminous lifetime is shortened, which makes it difficult to optically bomb titanium-sapphire in a flash lamp, and further shortens the service life of the lamp.
本発明では前記したチタンサファイアレーザーと非線形
光学材料を併用することで更に高精度の測定結果を得る
ことのできる装置とすることができる。In the present invention, by using the above-described titanium sapphire laser in combination with a nonlinear optical material, it is possible to create an apparatus that can obtain even more accurate measurement results.
ここで用いる非線形光学材料は、光の波長を変換するこ
とができる例えばβ−硼酸バリウム、ニオブ酸リチウム
などの無機質結晶、メタニトロアニリンなどの有機質結
晶等である。The nonlinear optical materials used here include, for example, inorganic crystals such as β-barium borate and lithium niobate, and organic crystals such as metanitroaniline, which can convert the wavelength of light.
次に本発明の構成を、本発明の一実施態様を例として説
明する。Next, the configuration of the present invention will be explained by taking one embodiment of the present invention as an example.
図１は本発明の一実施態様であるＩＩＰＬＣ用蛍光検用
益光検出器示す概略図である。図中１はチタンサファイ
アレーザー発振器、２はハーフミラ−３は試料セル、４
は偏光子、５は偏光解消板ンズ、６は集光レンズ、７は
分光器、８は検出器（例えば光電子増倍管）である。FIG. 1 is a schematic diagram showing a fluorescent light detector for IIPLC, which is an embodiment of the present invention. In the figure, 1 is a titanium sapphire laser oscillator, 2 is a half mirror, 3 is a sample cell, and 4
5 is a polarizer, 5 is a depolarizing lens, 6 is a condenser lens, 7 is a spectrometer, and 8 is a detector (for example, a photomultiplier tube).
本発明で用いる測定系の配置は、上記した配置に限定さ
れるものではなく、更に非線形光学材料はチタンサファ
イアレーザー発振器から試料にレーザー光を照射する間
に配置することができる。The arrangement of the measurement system used in the present invention is not limited to the arrangement described above, and the nonlinear optical material can be arranged while the sample is irradiated with laser light from the titanium sapphire laser oscillator.
本発明で、レーザー媒質であるチタンサファイア結晶を
レーザー発振させるための励起源としては、例えばフラ
ッシュランプ、ＹＡＧレーザーの２倍波、色素レーザー
、アルゴンレーザー、銅蒸気レーザ、発光ダイオード、
半導体レーザーなどがある。In the present invention, excitation sources for laser oscillation of the titanium sapphire crystal, which is a laser medium, include, for example, a flash lamp, a double wave of a YAG laser, a dye laser, an argon laser, a copper vapor laser, a light emitting diode,
Examples include semiconductor lasers.
また、波長のチューニングはプリズム、複屈折フィルタ
ー、回折格子、電気光学結晶、音響光学結晶などを用い
て行うことができる。Further, wavelength tuning can be performed using a prism, a birefringence filter, a diffraction grating, an electro-optic crystal, an acousto-optic crystal, etc.
図２は、フラッシュランプ励起によるレーザー発振器の
一実施態様の構成を示す概略図である。FIG. 2 is a schematic diagram showing the configuration of one embodiment of a flash lamp pumped laser oscillator.
図中９はチタンサファイアのロッド、ｌ０１ｌｌはフラ
ッシュランプ、１２．１３は反射ミラー、１４はプリズ
ム、１５はエタロン、１Ｇは楕円形の集光鏡である。In the figure, 9 is a titanium-sapphire rod, 1011 is a flash lamp, 12.13 is a reflecting mirror, 14 is a prism, 15 is an etalon, and 1G is an elliptical condenser mirror.
集光鏡の光反射面は銀の蒸着膜で構成すると、サファイ
アの励起光の反射効率が良く好ましい。It is preferable that the light reflecting surface of the condenser mirror be formed of a vapor-deposited silver film, since the reflection efficiency of the sapphire excitation light is good.
図３は、ＹＡＣの第２高周波で励起するサファイアレー
ザー発振器の一実施態様の構成を示す概略図である。図
中１７はＹＡＧレーザ−，１８は非線形光学材料の結晶
、１９は集光レンズ、２０．２１は反射ミラー、２２は
チタンサファイヤロッド、２３は複屈折フィルターであ
る。FIG. 3 is a schematic diagram showing the configuration of an embodiment of a sapphire laser oscillator excited with the second high frequency of YAC. In the figure, 17 is a YAG laser, 18 is a crystal of nonlinear optical material, 19 is a condenser lens, 20, 21 is a reflecting mirror, 22 is a titanium sapphire rod, and 23 is a birefringent filter.
上記の方法により、波長を約６９０〜１１０５０ｎの間
で連続的に変換することができる。The above method allows continuous wavelength conversion between approximately 690 and 11050 nm.
また、非線形光学材料を用い、第二高調波を発生させた
場合、約３４５〜５２５ｒｖの範囲内で波長を変換する
ことができる。また、第三高調波を発生させた場合、約
２３０〜３５０ｎｍの範囲内で波長を変換することがで
きる。Further, when a second harmonic is generated using a nonlinear optical material, the wavelength can be converted within the range of approximately 345 to 525 rv. Furthermore, when the third harmonic is generated, the wavelength can be converted within the range of about 230 to 350 nm.
［発明の効果］
上記のように、本発明はレーザー光の波長可変幅が広い
ことから、この範囲に吸収スペクトルを持つ物質は、レ
ーザー光の波長を吸収励起波長に合わせることが容易で
、蛍光標識しなくとも高感度で蛍光スペクトルの測定が
可能である。その結果、測定感度が向上し、低下濃度の
試料も充分測定可能となる。[Effects of the Invention] As described above, since the wavelength tunable range of laser light is wide in the present invention, substances with an absorption spectrum in this range can easily match the wavelength of the laser light to the absorption excitation wavelength, and can exhibit fluorescence. Fluorescence spectra can be measured with high sensitivity even without labeling. As a result, measurement sensitivity is improved, and even samples with reduced concentrations can be measured satisfactorily.
チタンサファイアレーザーは、連続発振、パルスのいず
れも可能で、ピコ秒パルスを利用すれば高時間分解の測
定が可能となる。Ti:sapphire lasers are capable of both continuous wave and pulsed lasers, and the use of picosecond pulses enables high time-resolved measurements.
本発明は、チタンサファイアレーザーを用い励起光を連
続的に変換可能であるので、蛍光標識することなく披７
１１１１定試料からの蛍光スペクトルを高選択的に高感
度でまた高時間分躬能で測定可能だあり、また安定かつ
容易にハ１定することができる。The present invention uses a titanium sapphire laser and can continuously convert excitation light, so it can be displayed without fluorescent labeling.
Fluorescence spectra from 1111 samples can be measured with high selectivity, high sensitivity, and high time resolution, and can be determined stably and easily.
さらに装置は簡易な構成で保守も簡便である。Furthermore, the device has a simple configuration and is easy to maintain.
図１は、本発明のＨＰＬＣ用蛍光検出器の一実施態様の
構成を示す概略図である。図中１はチタンサファイアレ
ーザー発振器、２はハーフミラ−３は試料セル、４は偏
光子、５は偏光解消板ンズ、６は集光レンズ、７は分光
器、８は検出器を夫々示す。
図２は、フラッシュランプ励起によるレーザー発振器の
一実施態様の構成を示す概略図である。
図中９はチタンサファイアのロッド、１０．１１はフラ
ッシュランプ、１２．１３は反射ミラー、１４はプリズ
ム、１５はエタロン、１６は楕円形の集光鏡を夫々示す
。
図３は、ＹＡＧの第２高周波で励起するサファイアレー
ザー発振器の一実施態様の構成を示す概略図である。図
中１７はＹＡＧレーザ−，１８は非線形光学材料の結晶
、１９は集光レンズ、２０．２１は反射ミラー、２２は
チタンサファイヤロッド、２３は複屈折フィルターを夫
々示す。FIG. 1 is a schematic diagram showing the configuration of one embodiment of the HPLC fluorescence detector of the present invention. In the figure, 1 is a titanium sapphire laser oscillator, 2 is a half mirror, 3 is a sample cell, 4 is a polarizer, 5 is a depolarizing lens, 6 is a condenser lens, 7 is a spectrometer, and 8 is a detector. FIG. 2 is a schematic diagram showing the configuration of one embodiment of a flash lamp pumped laser oscillator. In the figure, 9 is a titanium-sapphire rod, 10 and 11 are flash lamps, 12 and 13 are reflecting mirrors, 14 is a prism, 15 is an etalon, and 16 is an elliptical condenser mirror. FIG. 3 is a schematic diagram showing the configuration of an embodiment of a sapphire laser oscillator that excites YAG with a second high frequency. In the figure, 17 is a YAG laser, 18 is a crystal of a nonlinear optical material, 19 is a condenser lens, 20, 21 is a reflecting mirror, 22 is a titanium sapphire rod, and 23 is a birefringent filter.
Claims (1)
ーとして用いた高速液体クロマトグラフィー用蛍光検出
器 ２）波長可変固体レーザーが、チタンを添加したサファ
イア単結晶と非線形光学材料とを併用したものである特
許請求の範囲第１項記載の検出器[Claims] 1) Fluorescence detector for high performance liquid chromatography using titanium-doped sapphire as a wavelength tunable solid-state laser 2) A wavelength-tunable solid-state laser that uses a titanium-doped sapphire single crystal and a nonlinear optical material Detector according to claim 1, which is used in combination
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17720489A JPH0342550A (en) | 1989-07-11 | 1989-07-11 | Fluorescent detector for high-performance liquid chromatography |
| US07/547,359 US5037200A (en) | 1989-07-11 | 1990-07-03 | Laser-operated detector |
| GB9015163A GB2234852A (en) | 1989-07-11 | 1990-07-10 | Ti-doped sapphire laser-operated detector |
| DE4021955A DE4021955A1 (en) | 1989-07-11 | 1990-07-10 | LASER OPERATED DETECTING DEVICE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17720489A JPH0342550A (en) | 1989-07-11 | 1989-07-11 | Fluorescent detector for high-performance liquid chromatography |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0342550A true JPH0342550A (en) | 1991-02-22 |
Family
ID=16026996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17720489A Pending JPH0342550A (en) | 1989-07-11 | 1989-07-11 | Fluorescent detector for high-performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0342550A (en) |
-
1989
- 1989-07-11 JP JP17720489A patent/JPH0342550A/en active Pending
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