JPS58225351A - Analytical apparatus - Google Patents
Analytical apparatusInfo
- Publication number
- JPS58225351A JPS58225351A JP11018582A JP11018582A JPS58225351A JP S58225351 A JPS58225351 A JP S58225351A JP 11018582 A JP11018582 A JP 11018582A JP 11018582 A JP11018582 A JP 11018582A JP S58225351 A JPS58225351 A JP S58225351A
- Authority
- JP
- Japan
- Prior art keywords
- separation column
- column
- detector
- eluent
- component
- 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.)
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/84—Preparation of the fraction to be distributed
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】 本発明は分析装置に関するものである。[Detailed description of the invention] The present invention relates to an analysis device.
液体クロマトグラフ等の分離カラムが用いられた分析装
置により分析を行う場合、例えば血清中の胆汁酸、アン
ドロステロン、プルチゾール、プレグナンジオール、エ
ストロゲン等のステロイド系化合物やカテコールアミン
、ポリアミン等のアミンなどの非蛋白質成分や各種薬削
の血中濃度の定量にあたって、これらの成分を分離カラ
ム中で精度よく分離するために、溶離液中に特定の有機
溶媒や、塩類、界面活性剤、2−メルカプトエタノール
勢の添加剤などを加えることが行われているが、分離カ
ラムからの流出流を検出器にかけて該流出液中の分析目
的成分を検出する際に、溶離液中に分離をよくするため
に加えられた添加剤が該検出に悪影響を及はすことがあ
る。すなわち、流出流を固定化酵素カラムに通して、酵
素の作用によって分析目的物質を検出器例えば蛍光九度
肚による検出可能な物質に置き換えて検出を行う場合に
は、溶離液中の特定の有機溶媒が固定化酵素を失活させ
ることがあり、又、分析目的成分の紫外又は可視光の吸
収を利用して検出を行う場合には、溶離液中の添加成分
の吸収と分析目的成分の吸収が重なって検出しにくくな
ることがあり、又、分析目的成分と溶離液の屈折率の差
を利用して検出を行う場合KVi、溶離カラムにおける
分離向上のために添加された添加剤を含む溶離液の屈折
率が分析目的成分の屈折率に近づいて検出が困−となる
場合がある。When analysis is performed using an analyzer using a separation column such as a liquid chromatograph, for example, non-proteins such as bile acids in serum, steroid compounds such as androsterone, plutisol, pregnanediol, and estrogen, and amines such as catecholamines and polyamines are analyzed. In order to accurately separate these components in a separation column when quantifying blood concentrations of components and various drug substances, the eluent contains specific organic solvents, salts, surfactants, and 2-mercaptoethanol. Additives are added to the eluent to improve separation when the flow from the separation column is passed through a detector to detect the target component in the flow. Additives may adversely affect the detection. In other words, when the effluent is passed through an immobilized enzyme column and the target substance is replaced by a substance that can be detected by a detector, such as a fluorescence detector, by the action of the enzyme, the specific organic The solvent may deactivate the immobilized enzyme, and when detection is performed using ultraviolet or visible light absorption of the target component, the absorption of the added component in the eluent and the absorption of the target component In addition, when detecting using the difference in refractive index between the target component and the eluent, KVi, eluent containing additives added to improve separation in the elution column may overlap. There are cases where the refractive index of the liquid approaches the refractive index of the component to be analyzed, making detection difficult.
木発明けこの様な場合に、溶離液組成を変更することな
く、従って良好な分離精度を維持したオ\、分離カラム
よ秒後の検出系において溶離液中に含まれる検出阻害物
質によって検出精度を低下させるこさなく分析目的成分
を検出することの出来る分析装置を提供することを目的
き1、てなされたものであり、その要旨は、分離カラム
と検出器との間に、分離カラム流出液中に存在する分析
目的成分の検出に好ましくない成分を除去¥ftけ減少
せしめるための除去装置が設けられてなり、該除去装置
けその中を分離カラム流出液が流れる透析性を有する中
空管が、溶離液から上記好ましくない成分を除いた組成
の溶液中に浸漬されて々るものであるこ七を特徴とする
分析装置に存する。In a case like this, the separation column maintained good separation accuracy without changing the composition of the eluent, and the detection accuracy was reduced by the detection inhibitor contained in the eluent in the detection system seconds after the separation column. It was developed with the aim of providing an analyzer that can detect target components without reducing the A hollow tube having dialysis properties is provided, and a removal device is provided for removing and reducing components undesirable for detection of the analysis target component present in the column, and the separation column effluent flows through the removal device. The analyzer is immersed in a solution having a composition in which the above-mentioned undesirable components are removed from the eluent.
以下図面を参照しながら本発明装置について説明する。The apparatus of the present invention will be explained below with reference to the drawings.
vJ1図は本発明装置の一実施例を示す系統図である。Figure vJ1 is a system diagram showing one embodiment of the device of the present invention.
図中1は溶離液槽であり、該槽1中の溶離液がポンプ2
により、r、試料インジェクター3、分離カラム4、除
去装置5、固定化酵素カラム8及び検出器9を通過する
ように流される七同時に、反応液槽6中の反応液がポン
プ7によ沙、固定化酵素カラム80手前で溶離液流と合
流されるように々されている。該図に示される様な反応
液及び固定化酵素力う八8が用いられる分析システムは
、分析目的成分が検出器例えば蛍光光度計等に対して活
性を有さす、分離カラム4によって分離されてもそのま
\では検出器9で検出され得ない場合罠採用されるシス
テムであり、分離カラム4かもの流出液を同定化酵素カ
ラム8に導き、ここで酵素の作用により、分析目的成分
とすでに合流させた反応液中に含まれる反応物質とを反
応させて、分析目的成分を検出可能な物質に変質せしめ
るか若しく打分析目的成分の量に対応する量の検出可能
な物質を ′□生成せしめることによ秒、検出器
9によって分析目的成分の定量が可能となされたシステ
ムである。又、10#′i検出器9による検出を記録す
るための記録11である。1 in the figure is an eluent tank, and the eluent in tank 1 is pumped 2.
At the same time, the reaction solution in the reaction solution tank 6 is passed through the sample injector 3, the separation column 4, the removal device 5, the immobilized enzyme column 8, and the detector 9. It is arranged to be combined with the eluent stream before the immobilized enzyme column 80. In an analysis system in which a reaction solution and an immobilized enzyme 8 as shown in the figure are used, the components to be analyzed are separated by a separation column 4 that is active for a detector such as a fluorometer. This is a system that is used when the sample cannot be detected by the detector 9.The effluent from the separation column 4 is led to the identification enzyme column 8, where it is already separated from the target component by the action of the enzyme. By reacting with the reactant contained in the combined reaction solution, the target component for analysis is transformed into a detectable substance, or a detectable substance is generated in an amount corresponding to the amount of the target component for analysis. This system makes it possible to quantify the component to be analyzed using the detector 9 in seconds. It is also a record 11 for recording the detection by the 10#'i detector 9.
上記の如き分析システムが用いられる分析目的成分の一
例として胆汁酸の各成分を挙けることが出来、この場合
、酵素3α−I(SD(3α−ヒドロキシステロイドデ
ヒドロゲナーゼ)が固定化された固定化酵素カラム、N
AD+にコチン酸アミドアデニンジヌクレオチド)を含
む反応液及び検出器として蛍光光度計を用い、固定化酵
素カラム中で30+−1(SDの作用により胆汁酸成分
とNAD とを反応させ、その結果、反応した胆汁酸
と等量生成する蛍光物質NAD)1を検出器によって検
出することにより分析を行うことが出来るのである。An example of a component to be analyzed using the above-mentioned analysis system is each component of bile acids. Column, N
Using a reaction solution containing AD+ (cotinamide adenine dinucleotide) and a fluorometer as a detector, the bile acid component and NAD were reacted by the action of 30+-1 (SD) in an immobilized enzyme column, and as a result, Analysis can be performed by using a detector to detect the fluorescent substance NAD) 1 produced in an equal amount to the reacted bile acid.
しかしながら、一般に固定化酵素カラムは、分離カラム
における分離精度を向上させるために必要な溶離液中の
有機溶剤例えば胆汁酸分析におけるアセトニトリル等罠
よってその酵素活性が損われ、長時間の分析が困難とな
る場合が多く、本発明においては、この様な、分離カラ
ム4による分離では必要であったがそれ以後の分析目的
成分の検出においては好ましくない成分ム
を除1tけ減少せしめるために、除去装fft5が分離
カラム4と検出器8との同に設けられるのである。そし
て、該除去装置5け、透析性を有する中空管51が、検
出において好ましくないとされる除去目的成分を溶離液
組成から除いた組成の溶液中に浸漬された構造のもので
あり、接続端52.53により分析システム系に接続さ
れて上記中空管51中を分離カラム4からの流出液が通
過する様になされている。そして上記流出液が中空管5
1を通過する間に透析の原理に従って、溶離液には含ま
れるが上記溶液中には含まれない成分すなわち除去目的
成分は中空管51の管壁から上記溶液中に浸透し散逸す
るのであり、それによって中空管51を流れる分離カラ
ム流出液から除去目的成分が除去或は減少せしめられる
のである。しかして中空管51としては、除去目的成分
に対して透析性を有するものが使用されるが、さらに当
然のことながらこれ罠接触する溶液に侵されないこと、
システムiKかけられる内圧に耐えられること及び分析
目的物質に対し不活性で吸着したりしないものであるこ
とが要求される。又、該中空管51として透析性を有す
る細管を複数本束ねたものを用いることも可能である。However, in general, the enzyme activity of immobilized enzyme columns is impaired by organic solvents in the eluent, such as acetonitrile in bile acid analysis, which are necessary to improve the separation accuracy in the separation column, making long-term analysis difficult. In the present invention, in order to reduce by 1 ton of such components that are necessary for separation using the separation column 4 but are undesirable for subsequent detection of the target component, a removal device is used. fft5 is provided at the same time as the separation column 4 and the detector 8. The five removal devices have a structure in which a hollow tube 51 having dialysis properties is immersed in a solution having a composition in which the target component to be removed, which is considered undesirable in detection, is removed from the eluent composition. The ends 52 and 53 are connected to the analysis system so that the effluent from the separation column 4 passes through the hollow tube 51. Then, the above-mentioned effluent flows into the hollow tube 5
According to the principle of dialysis, components contained in the eluent but not contained in the solution, that is, the target components to be removed, permeate into the solution from the wall of the hollow tube 51 and dissipate. As a result, the component to be removed is removed or reduced from the separation column effluent flowing through the hollow tube 51. Therefore, the hollow tube 51 used is one that has dialysis properties against the target component to be removed, but it also needs to be protected from being attacked by the solution it comes into contact with.
It is required to be able to withstand the internal pressure applied to the system iK, and to be inert and not adsorbed to the substance to be analyzed. Further, it is also possible to use a bundle of a plurality of thin tubes having dialysis properties as the hollow tube 51.
又、該中空管51としては透析性と同時に除去目的成分
に対して限外ri過性を有するものも使用可能である。Further, as the hollow tube 51, it is also possible to use a tube that has both dialysis properties and ultra-RI permeability for the component to be removed.
本発明に用いられて好適な中空管51きしては、再生セ
ルロース、酢酸セルロース、ポリアクリロニトリル、ポ
リスルホン、ポリプロピレン等高分子材料から製せられ
た透析性を有する管状体が挙けられ、人工腎臓用透析膜
として市販されている細管を用いることも可能である。The hollow tube 51 suitable for use in the present invention includes a dialyzable tubular body made of a polymeric material such as regenerated cellulose, cellulose acetate, polyacrylonitrile, polysulfone, or polypropylene. It is also possible to use thin tubes commercially available as kidney dialysis membranes.
以しにおいて、胆汁酸等の分析の如くに固定化酵素カラ
ムが用いられる場合について説明したが、本発明装置け
これに限られる如く、固定化酵素カラムが用いられない
場合でも、溶離液中に含まれる成分の光吸収や屈折率な
どが分析目的成分のそれと近似して検出器による分析目
的成分の検出が困難てあったり、その他溶離液中の成分
が分析目的成分の検出の段階で好ましくない挙動を示し
て、該溶離液中の成分を除去することが分析に好結果を
与える場合には採用されることが出来、すぐれた効果が
期待出来る。In the following, we have explained the case where an immobilized enzyme column is used, such as in the analysis of bile acids, etc. However, even when an immobilized enzyme column is not used, as is limited to the device of the present invention, it is possible to The light absorption or refractive index of the contained components is similar to that of the target component, making it difficult for the detector to detect the target component, or other components in the eluent are undesirable at the stage of detecting the target component. If removing the components in the eluent by showing the behavior of the eluent gives good results in the analysis, it can be adopted, and excellent effects can be expected.
本発明は上述の通りの装置であり、とくに分離カラムと
検出器との間に、該分離カラム流出液中に含まれる分離
カラムによる分析目的成分の分離では必要であったが、
それよシ後の分析目的成分の検出の段階では存在するこ
七が好オしくない成分を除去するための特定構造の除去
装置が設けられてなるものであるから、該好ましくない
成分を有効に除去して検出における該成分の悪影響を取
り除き、それ罠よって例えば分析の精度を向上させたり
、又は検出系における検出能力の低下を防止して長時間
の連続分析を可能ならしめるとhったすぐれた効果を奏
し得るのである。The present invention is an apparatus as described above, and in particular, between the separation column and the detector, it is necessary to separate the target component for analysis by the separation column contained in the separation column effluent.
In addition, in the subsequent stage of detection of the components to be analyzed, a removal device with a specific structure is installed to remove the undesirable components present, so that the undesirable components can be effectively removed. It is excellent to eliminate the adverse effects of the component on detection by removing it, for example, to improve the accuracy of analysis, or to prevent a decline in the detection ability of the detection system and enable long-term continuous analysis. Therefore, it can have a great effect.
次に本発明の実施例について説明する。Next, examples of the present invention will be described.
実施例1
第1図に示される装置を用いてヒト血清中より抽出した
胆汁酸の分析を繰返して行った。Example 1 Bile acids extracted from human serum were repeatedly analyzed using the apparatus shown in FIG.
ここで分離カラム4としてはクオーターズ社製マイクロ
ポンダノ(ツクフェニル(商品名)を用い、検出器9と
してij蛍光計を用いて励起波長365nm、蛍光波長
450nmで測定を行った。Here, Micropondano (trade name) manufactured by Quarters Co., Ltd. was used as the separation column 4, and an IJ fluorometer was used as the detector 9, and measurement was performed at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm.
又、除去装置5としては、再生セルロース製ノ人工腎臓
用中空糸(西独Enka社Ilりを2mの長さに切断し
たもの5木を束ねたものが中空管6として用いられ、こ
れがll中に15yのリン酸2アンモニクムを含む水溶
液に浸漬された構造のものが用いられた。又、固定化酵
素カラム8としては、セルロース微粒子(a径約80μ
)に酵素3α−)ISDが、次酸ナトリクム水溶H中ノ
(! tv a−ス微粒子にシアン化プf7fイドが溶
解されたアセトニトリルを加えて該微粒子表面を活性化
させ、これに3α−ISDを溶解させた炭酸緩衝液を加
えて撹拌する方法により固定化された3α−HS D固
定化セルロース微粒子が、長さ100m、内径4flの
カラムに充填されたものを用いた。As the removal device 5, a hollow fiber for artificial kidneys made of regenerated cellulose (manufactured by Enka Co., West Germany, cut into a length of 2 m) 5 made of wood bundled together is used as a hollow tube 6. For the immobilized enzyme column 8, cellulose fine particles (a diameter of about 80 μ
), the enzyme 3α-ISD is added to the acetonitrile in which cyanide pseudo-f7f is dissolved in sodium subacid aqueous solution H (! tv a-s) to activate the surface of the microparticles, and 3α-ISD is added to the microparticles. A column having a length of 100 m and an inner diameter of 4 fl was filled with 3α-HSD-immobilized cellulose fine particles that had been immobilized by adding and stirring a carbonate buffer solution in which 3α-HSD was dissolved.
11!中に300 meのアセトニトリルとL59のリ
ン酸2アンモニウムを含む水溶液を溶離液として用い、
ポンプ2により該溶離液を1m//分の流速で溶離液槽
1から分離カラム4、除去装置5、固定化酵素カラム8
及び検出器9を迎渦する様に流し、又、反応液槽6中の
ll中にNAD 199.1■及びエチレンジアミン
4酢酸24.5 fnqを含む0.026 Mビロリン
酸緩lrJ液(P H9,5)からなる反応液をポンプ
7により111127分の流速で、固定化酵素カラム8
に流入する前の溶離液流と合流させた。11! Using an aqueous solution containing 300 me acetonitrile and L59 diammonium phosphate as an eluent,
The eluent is transferred from the eluent tank 1 to the separation column 4, the removal device 5, and the immobilized enzyme column 8 at a flow rate of 1 m/min by the pump 2.
A 0.026 M birophosphoric acid mild lrJ solution (P H9 , 5) at a flow rate of 111,127 min by pump 7 to the immobilized enzyme column 8
The eluent stream was then merged with the eluent stream before entering.
ここで試料インジェクター3から前記胆汁酸試料を注入
すると、分離カラム4において分画さされた。そ[2て
この分析を500時間繰りyi、t。When the bile acid sample was injected from the sample injector 3, it was fractionated in the separation column 4. Then, repeat the analysis for 500 hours yi, t.
て行っても、同定化酵素カラム8の溶Nt液中(で含ま
れるアセトニトリルにょる失活り殆んど起らず、最初と
同様な活性を示した。そして、溶離液中のアセトニトリ
ルは、除去装置5を通過17だときにその約90%が除
去されることが確認された。Even when it was carried out, there was almost no inactivation due to the acetonitrile contained in the eluted Nt solution of identification enzyme column 8, and the same activity as the first one was shown. It was confirmed that about 90% of the particles were removed when the particles passed through the removal device 5 (17).
比較例1
除去袋WII5を使用しないこと以外Fip施例1と同
様にして、胆汁酸の分析を繰り返して連続的に行ったと
ころ、溶離液中のアセトニトリルによって同定化酵素が
次第に失活し、50時間後Comparative Example 1 Bile acid analysis was continuously repeated in the same manner as in Fip Example 1 except that the removal bag WII5 was not used. The identified enzyme was gradually inactivated by acetonitrile in the eluent, and After hours
第1図は本発明装置の一例を示す系統図である。
1・・・W#離液槽、2.7・・・ポンプ、3・・・試
料インジェクター、4・・・分離カラム、5・・・除去
装置、6・・・透析性を有する中空管、6・・・反応液
槽、8・・・固定化酵素カラム、9・・・検出器、10
・・・記鐙計
特許出願人 積水化学工業株式会社
代表者 藤 沼 基 利FIG. 1 is a system diagram showing an example of the apparatus of the present invention. 1...W# syneresis tank, 2.7...pump, 3...sample injector, 4...separation column, 5...removal device, 6...hollow tube with dialysis properties , 6... Reaction liquid tank, 8... Immobilized enzyme column, 9... Detector, 10
...Stirrup meter patent applicant Mototoshi Fujinuma, representative of Sekisui Chemical Co., Ltd.
Claims (1)
に存在する分析目的成分の検出に好ましくない成分を除
去或は減少せしめるための除去装置が設けられてなり、
該除去装置はその中を分離カラム流出液が流れる透析性
を有する中空管が、溶離液から上記好ましくない成分を
除いた組成の溶液中に浸漬されてなるものであることを
特徴とする分析装置。t. A removal device is provided between the separation column and the detector to remove or reduce components that are present in the separation column effluent and are undesirable for detection of the analysis target component,
The removal device is characterized in that a hollow tube having dialysis properties through which the separation column effluent flows is immersed in a solution having a composition in which the above-mentioned undesirable components are removed from the eluent. Device.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP11018582A JPS58225351A (en) | 1982-06-25 | 1982-06-25 | Analytical apparatus |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP11018582A JPS58225351A (en) | 1982-06-25 | 1982-06-25 | Analytical apparatus |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS58225351A true JPS58225351A (en) | 1983-12-27 |
Family
ID=14529194
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP11018582A Pending JPS58225351A (en) | 1982-06-25 | 1982-06-25 | Analytical apparatus |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS58225351A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2019501396A (en) * | 2015-11-20 | 2019-01-17 | アクティベイティッド リサーチ カンパニー、エルエルシー | Catalytic reactor for liquid chromatography coupled to a flame ionization detector |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS53113597A (en) * | 1977-03-16 | 1978-10-04 | Hitachi Ltd | Liquid chromatography |
JPS56135156A (en) * | 1980-01-16 | 1981-10-22 | Dow Chemical Co | Chromatograph analyzer |
-
1982
- 1982-06-25 JP JP11018582A patent/JPS58225351A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS53113597A (en) * | 1977-03-16 | 1978-10-04 | Hitachi Ltd | Liquid chromatography |
JPS56135156A (en) * | 1980-01-16 | 1981-10-22 | Dow Chemical Co | Chromatograph analyzer |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2019501396A (en) * | 2015-11-20 | 2019-01-17 | アクティベイティッド リサーチ カンパニー、エルエルシー | Catalytic reactor for liquid chromatography coupled to a flame ionization detector |
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