JP6977863B2 - Quinone analysis method and online SFE-SFC system for implementing the method - Google Patents

Quinone analysis method and online SFE-SFC system for implementing the method Download PDF

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JP6977863B2
JP6977863B2 JP2020502033A JP2020502033A JP6977863B2 JP 6977863 B2 JP6977863 B2 JP 6977863B2 JP 2020502033 A JP2020502033 A JP 2020502033A JP 2020502033 A JP2020502033 A JP 2020502033A JP 6977863 B2 JP6977863 B2 JP 6977863B2
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恵子 松本
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/40—Selective adsorption, e.g. chromatography characterised by the separation mechanism using supercritical fluid as mobile phase or eluent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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    • B01J20/287—Non-polar phases; Reversed phases
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    • 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
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    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Description

本発明は、キノン類分析方法とその方法を実施するためのオンラインＳＦＥ−ＳＦＣシステムに関するものである。 The present invention relates to a quinone analysis method and an online SFE-SFC system for carrying out the method.

土壌などの試料中における微生物の活動を観察するための手法の１つとしてキノンプロファイリング法がある（特許文献１を参照。）。キノンプロファイリング法は、微生物の呼吸関連物質であるキノン類を試料から抽出してその量を測定することで、試料中に存在する微生物群集の種類や量を把握する手法である。 There is a quinone profiling method as one of the methods for observing the activity of microorganisms in a sample such as soil (see Patent Document 1). The quinone profiling method is a method for grasping the type and amount of microbial communities present in a sample by extracting quinones, which are microbial respiration-related substances, from the sample and measuring the amount thereof.

キノン類の抽出には、一般的に有機溶媒による手動抽出の他に、超臨界流体抽出（ＳＦＥ）システムが利用される。具体的には、試料を封入した抽出容器に超臨界状態の二酸化炭素と適量の有機溶媒からなる移動相溶媒を通液することによって抽出容器内の試料からキノン類を抽出し、試料から抽出されたキノン類を吸着カートリッジに捕捉させる。 For extraction of quinones, a supercritical fluid extraction (SFE) system is generally used in addition to manual extraction with an organic solvent. Specifically, quinones are extracted from the sample in the extraction container by passing a mobile phase solvent consisting of supercritical carbon dioxide and an appropriate amount of organic solvent through the extraction container containing the sample, and the sample is extracted from the sample. The quinones are captured by the adsorption cartridge.

試料からキノン類を抽出した後、キノン類を捕捉させた吸着カートリッジをＳＦＥシステムから取り外し、その吸着カートリッジにアセトン溶液を通液することによって吸着カートリッジからキノン類を溶出させる。溶出したキノン類を含有するアセトン溶液を別のシリカゲルカラムに通液し、シリカゲルカラムにキノン類を吸着させる。そして、キノン類を吸着させたシリカゲルカラムに揮発性の高い有機溶媒を通液してキノン類を溶出させて回収した後、回収した液をエバポレータで蒸発させて濃縮し、アセトンで洗浄してキノン類を回収し、回収したキノン類を高速液体クロマトグラフ（ＨＰＬＣ）にて分析することにより定量する。 After extracting the quinones from the sample, the adsorption cartridge that has captured the quinones is removed from the SFE system, and the quinones are eluted from the adsorption cartridge by passing an acetone solution through the adsorption cartridge. An acetone solution containing the eluted quinones is passed through another silica gel column, and the quinones are adsorbed on the silica gel column. Then, a highly volatile organic solvent is passed through a silica gel column on which quinones are adsorbed to elute and recover the quinones, and then the recovered liquid is evaporated by an evaporator to concentrate, washed with acetone and washed with quinone. The species are collected and the recovered quinones are quantified by analysis by high performance liquid chromatography (HPLC).

ＷＯ２００６／１１８１６７Ａ１WO2006 / 118167A1 ＷＯ２０１６／０３１００８Ａ１WO2016 / 0310008A1

上記のように、従来のキノンプロファイリング法では、ＳＦＥシステムにて試料からキノン類を抽出した後、抽出されたキノン類をＨＰＬＣに導入するまでの脱離や濃縮といった処理を手作業で行なう必要があった。 As described above, in the conventional quinone profiling method, it is necessary to manually perform processes such as desorption and concentration after extracting quinones from a sample by an SFE system and then introducing the extracted quinones into HPLC. there were.

本発明は、キノン類の抽出から分析までを手作業を介することなく高効率に行なえるようにすることを目的とするものである。 An object of the present invention is to enable high efficiency from extraction to analysis of quinones without manual labor.

本発明に係るキノン類分析方法は、キノン類の抽出から分離分析までをオンラインＳＦＥ−ＳＦＣシステムを用いて手作業を介することなく実行するものである。 The quinone analysis method according to the present invention is to perform from extraction of quinones to separation analysis using an online SFE-SFC system without manual intervention.

オンラインＳＦＥ−ＳＦＣシステムは、超臨界流体を用いた試料成分の抽出（ＳＦＥ）と、抽出された成分の超臨界流体クロマトグラフィー分析（ＳＦＣ）とをオンラインで行なうことができるように構成されたシステムであり、例えば特許文献２に開示されている。具体的には、超臨界状態の二酸化炭素とモディファイアからなる移動相溶媒を供給するための移動相供給部、試料が封入され、前記移動相供給部から供給される前記移動相溶媒によって前記試料から成分を抽出するための抽出容器、前記試料から抽出された成分の分離を行なうための分析カラム、及び前記分析カラムで分離された成分を検出するための検出器を有するものであり、前記抽出容器内で試料中に含まれる成分を前記移動相溶媒に抽出した後、前記試料から抽出された成分を含む前記移動相溶媒を前記分析カラムに導いて分離し、前記分析カラムで分離された成分を前記検出器により検出するように構成されたものである。 The online SFE-SFC system is a system configured to enable extraction of sample components using supercritical fluid (SFE) and supercritical fluid chromatography analysis (SFC) of the extracted components online. For example, it is disclosed in Patent Document 2. Specifically, a mobile phase supply unit and a sample for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state are enclosed, and the sample is supplied by the mobile phase solvent supplied from the mobile phase supply unit. It has an extraction container for extracting components from the sample, an analysis column for separating the components extracted from the sample, and a detector for detecting the components separated by the analysis column. After the components contained in the sample were extracted into the mobile phase solvent in the container, the mobile phase solvent containing the components extracted from the sample was guided to the analysis column and separated, and the components separated by the analysis column were separated. Is configured to be detected by the detector.

本発明のキノン類分析方法は、前記抽出容器にキノン類を含む試料を封入する試料封入ステップと、前記試料封入ステップの後で、前記移動相供給部から前記試料からキノン類を含む成分を抽出するための第１の移動相溶媒を前記抽出容器に供給し、前記抽出容器内が前記第１の移動相溶媒で満たされた状態で静置する「静的抽出」を実施した後、前記試料から抽出された成分を含む前記第１の移動相溶媒を前記分析カラムに通液し、前記第１の移動相溶媒に含まれる成分のうち少なくともキノン類を含む成分を前記分析カラムに捕捉させる「動的抽出」を一連の抽出動作として実行する抽出ステップと、前記一連の抽出動作により前記分析カラムに捕捉された成分を前記分析カラムから溶出させるための第２の移動相溶媒を前記移動相供給部から供給して前記分析カラムに通液し、前記分析カラムに捕捉された成分を分離しながら溶出させて前記検出器により検出する分析ステップと、を備えている。 In the quinone analysis method of the present invention, a sample encapsulation step of encapsulating a sample containing quinone in the extraction container, and after the sample encapsulation step, a component containing quinone is extracted from the sample from the mobile phase supply unit. After performing "static extraction" in which the first mobile phase solvent for this purpose is supplied to the extraction container and the inside of the extraction container is filled with the first mobile phase solvent and allowed to stand, the sample is prepared. The first mobile phase solvent containing the components extracted from the above is passed through the analysis column, and the components containing at least quinones among the components contained in the first mobile phase solvent are captured in the analysis column. A mobile phase supply of an extraction step in which "dynamic extraction" is performed as a series of extraction operations and a second mobile phase solvent for eluting the components captured in the analysis column by the series of extraction operations from the analysis column. It is provided with an analysis step of supplying a liquid from a section, passing the liquid through the analysis column, eluting the components captured in the analysis column while separating them, and detecting the components by the detector.

本発明のキノン類分析方法では、前記抽出ステップにおいて「静的抽出」と「動的抽出」からなる前記一連の抽出動作を複数回、好ましくは３回以上繰り返すことが好ましい。そうすれば、キノン類の抽出率が向上し、分析精度を高めることができる。 In the quinone analysis method of the present invention, it is preferable to repeat the series of extraction operations including "static extraction" and "dynamic extraction" a plurality of times, preferably three times or more in the extraction step. Then, the extraction rate of quinones can be improved and the analysis accuracy can be improved.

前記第２の移動相溶媒として、前記モディファイアの濃度が前記第１の移動相溶媒よりも高いものを用いることができる。 As the second mobile phase solvent, one having a higher concentration of the modifier than the first mobile phase solvent can be used.

前記分析ステップにおいて、前記第２の移動相溶媒の前記モディファイアの濃度を時間とともに上昇させてもよい。 In the analysis step, the concentration of the modifier of the second mobile phase solvent may be increased over time.

本発明に係るオンラインＳＦＥ−ＳＦＣシステムは、上述のキノン類分析方法を全自動で実施する機能を備えるものである。具体的には、当該オンラインＳＦＥ−ＳＦＣシステムの動作制御を行なう制御部が、キノン類を含む試料が封入された前記抽出容器に、前記試料からキノン類を含む成分を抽出するための第１の移動相溶媒を供給して前記抽出容器を前記第１の移動相溶媒で満たして静置した後、前記試料から抽出された成分を含む前記抽出容器内の前記第１の移動相溶媒を前記分析カラムに通液し、前記第１の移動相溶媒中の成分のうち少なくともキノン類を含む成分を前記分析カラムに捕捉させる一連の抽出動作を実行するように構成された抽出部と、前記一連の抽出動作により前記分析カラムに捕捉された成分を前記分析カラムから溶出させるための前記第２の移動相溶媒を前記分析カラムに通液するように構成された分析部と、を備えている。 The online SFE-SFC system according to the present invention has a function of fully automatically performing the above-mentioned quinone analysis method. Specifically, the control unit that controls the operation of the online SFE-SFC system is the first for extracting a component containing quinone from the sample into the extraction container in which the sample containing quinone is enclosed. After supplying the mobile phase solvent and filling the extraction container with the first mobile phase solvent and allowing it to stand, the first mobile phase solvent in the extraction container containing the components extracted from the sample is analyzed. An extraction unit configured to carry out a series of extraction operations for passing a liquid through the column and causing the analysis column to capture at least a component containing quinones among the components in the first mobile phase solvent, and the series. It is provided with an analysis unit configured to pass the second mobile phase solvent for eluting the components captured in the analysis column by the extraction operation from the analysis column.

前記抽出部は、前記一連の抽出動作を複数回繰り返すように構成されていることが好ましい。そうすれば、キノン類の抽出率が向上し、分析精度を高めることができる。 The extraction unit is preferably configured to repeat the series of extraction operations a plurality of times. Then, the extraction rate of quinones can be improved and the analysis accuracy can be improved.

本発明を実現するための前記分析カラムとしては、担体にナフチルエチル基又はピレニルエチル基を結合させた分離媒体が充填されているナフチルエチル基又はピレニルエチル基結合型のカラムが挙げられ、前記モディファイアとしてアセトニトリルが挙げられる。 Examples of the analytical column for realizing the present invention include a naphthylethyl group or pyrenylethyl group-bonded column in which a separation medium in which a naphthylethyl group or a pyrenylethyl group is bonded to a carrier is filled, and the modifier thereof includes a naphthylethyl group or pyrenylethyl group-bound column. Acetonitrile can be mentioned.

本発明のキノン類分析方法では、分析者が抽出容器にキノン類を含む試料を封入した後、第１の移動相溶媒で抽出容器を満たした状態で静置しキノン類を含む成分を前記第１の移動相溶媒に抽出する静的抽出、及び前記抽出容器内の前記第１の移動相溶媒を前記分析カラムに通液してキノン類を含む成分を前記分析カラムに捕捉させる動的抽出を一連の抽出動作として実施し、その後、第２の移動相溶媒を前記分析カラムに通液して前記分析カラムに捕捉された成分を分離しながら溶出させて前記検出器により検出するので、試料中のキノン類の抽出から分離分析までを１つのＳＦＥ−ＳＦＣシステム上で完結させることができる。 In the quinone analysis method of the present invention, after the analyst encloses a sample containing quinone in an extraction container, the extraction container is filled with the first mobile phase solvent and allowed to stand to contain the component containing quinone. Static extraction to be extracted into the mobile phase solvent of 1, and dynamic extraction in which the first mobile phase solvent in the extraction container is passed through the analysis column to capture components containing quinones in the analysis column. It is carried out as a series of extraction operations, and then a second mobile phase solvent is passed through the analysis column to elute the components captured in the analysis column while separating them, and the components are detected by the detector. From the extraction of quinones to the separation analysis can be completed on one SFE-SFC system.

本発明のオンラインＳＦＥ−ＳＦＣシステムでは、上記キノン類分析方法を全自動で実施する機能を有するので、キノン類の抽出から分離分析までの処理を高効率に行なうことができる。 Since the online SFE-SFC system of the present invention has a function of fully automatically performing the above-mentioned quinone analysis method, it is possible to perform processing from extraction of quinones to separation analysis with high efficiency.

オンラインＳＦＥ−ＳＦＣシステムの一実施例を概略的に示す流路構成図である。It is a flow path block diagram schematically showing an embodiment of an online SFE-SFC system. 同実施例のオンラインＳＦＥ−ＳＦＣシステムを用いたキノン類の分析方法を説明するためのフローチャートである。It is a flowchart for demonstrating the analysis method of quinones using the online SFE-SFC system of the same Example. スタンバイ時のオンラインＳＦＥ−ＳＦＣシステムの流路構成を示す図である。It is a figure which shows the flow path composition of the online SFE-SFC system at the time of standby. 静的抽出時のオンラインＳＦＥ−ＳＦＣシステムの流路構成を示す図である。It is a figure which shows the flow path composition of the online SFE-SFC system at the time of static extraction. 動的抽出時のオンラインＳＦＥ−ＳＦＣシステムの流路構成を示す図である。It is a figure which shows the flow path composition of the online SFE-SFC system at the time of dynamic extraction. 分析時のオンラインＳＦＥ−ＳＦＣシステムの流路構成を示す図である。It is a figure which shows the flow path composition of the online SFE-SFC system at the time of analysis. 同実施例のオンラインＳＦＥ−ＳＦＣシステムを用いたキノン類の分析方法による分析結果の一例を示すクロマトグラムであり、（Ａ）は浄化前の土壌の分析結果、（Ｂ）は浄化後の土壌の分析結果である。It is a chromatogram showing an example of the analysis result by the analysis method of quinones using the online SFE-SFC system of the same example, (A) is the analysis result of the soil before purification, and (B) is the soil after purification. It is an analysis result. 一連の抽出動作の繰り返し回数によるキノン類の抽出効果の検証結果を示すグラフであり、（Ａ）は繰り返し回数が１回の場合、（Ｂ）は繰り返し回数が３回の場合である。It is a graph which shows the verification result of the extraction effect of quinones by the number of repetitions of a series of extraction operations, (A) is the case where the number of repetitions is 1, and (B) is the case where the number of repetitions is three.

以下、本発明に係るキノン類分析方法及びその方法を実施するためのオンラインＳＦＥ−ＳＦＣシステムについて、図面を参照しながら説明する。 Hereinafter, the quinone analysis method according to the present invention and the online SFE-SFC system for carrying out the method will be described with reference to the drawings.

図１に、キノン類分析方法の実施に適したオンラインＳＦＥ−ＳＦＣシステムの一実施例の概略構成図を示す。 FIG. 1 shows a schematic configuration diagram of an embodiment of an online SFE-SFC system suitable for carrying out a quinone analysis method.

この実施例のオンラインＳＦＥ−ＳＦＣシステム１は、主として、移動相供給部２、ＳＦＥ部４、オートサンプラ６、分析カラム８、背圧制御バルブ（ＢＰＲ）１０，１２、検出器１４、及び制御部２８からなる。 The online SFE-SFC system 1 of this embodiment mainly includes a mobile phase supply unit 2, an SFE unit 4, an autosampler 6, an analysis column 8, back pressure control valves (BPR) 10, 12, a detector 14, and a control unit. It consists of 28.

移動相供給部２は、二酸化炭素を送液するための二酸化炭素ポンプ１６と、モディファイアを送液するためのモディファイアポンプ１８を備えている。二酸化炭素の初期状態は液体であるが、ＢＰＲ１０又は１２によって流路内の圧力が制御されることにより、流路中を超臨界状態で流れる。モディファイアとしては、例えばアセトニトリルが挙げられる。 The mobile phase supply unit 2 includes a carbon dioxide pump 16 for sending carbon dioxide and a modifier pump 18 for sending a modifier. Although the initial state of carbon dioxide is a liquid, it flows in a supercritical state in the flow path by controlling the pressure in the flow path by BPR 10 or 12. Examples of the modifier include acetonitrile.

ＳＦＥ部４は、抽出容器２０、流路切替バルブ２２及び２４を備えている。流路切替バルブ２２及び２４はそれぞれ、抽出容器２０の一端及び他端の接続先を切り替えるためのものである。流路切替バルブ２２及び２４の切替えにより、このオンラインＳＦＥ−ＳＦＣシステム１は、スタンバイ状態（図３参照）のほか、静的抽出（図４参照）、動的抽出（図５参照）及び分析（図６参照）のそれぞれの動作を実施するための状態をとることができる。静的抽出（図４参照）、動的抽出（図５参照）及び分析（図６参照）の詳細については後述する。 The SFE unit 4 includes an extraction container 20, a flow path switching valve 22 and 24. The flow path switching valves 22 and 24 are for switching the connection destinations of one end and the other end of the extraction container 20, respectively. By switching the flow path switching valves 22 and 24, the online SFE-SFC system 1 is in a standby state (see FIG. 3), as well as static extraction (see FIG. 4), dynamic extraction (see FIG. 5) and analysis (see FIG. 5). It is possible to take a state for carrying out each operation of (see FIG. 6). Details of static extraction (see FIG. 4), dynamic extraction (see FIG. 5) and analysis (see FIG. 6) will be described later.

オートサンプラ６は移動相供給部２からの移動相溶媒が流れる流路上におけるＳＦＥ部４の下流に設けられている。オートサンプラ６は、例えば、標準試料の測定データを取得する際に流路中に標準試料を注入するためのものである。 The autosampler 6 is provided downstream of the SFE unit 4 on the flow path through which the mobile phase solvent from the mobile phase supply unit 2 flows. The autosampler 6 is for injecting a standard sample into a flow path, for example, when acquiring measurement data of the standard sample.

分析カラム８はオートサンプラ６のさらに下流に設けられている。分析カラム８は、例えば、シリカゲル担体にナフチルエチル基を結合させた分離媒体が充填されたナフチルエチル基結合型のカラムである。分析カラム８はカラムオーブン２６内に収容され、温度調節がなされる。 The analysis column 8 is provided further downstream of the autosampler 6. The analysis column 8 is, for example, a naphthylethyl group-bonded column packed with a separation medium in which a naphthylethyl group is bound to a silica gel carrier. The analysis column 8 is housed in a column oven 26 and is temperature controlled.

ＢＰＲ１０はオートサンプラ６と分析カラム８との間の流路から分岐した流路上に設けられており、ＢＰＲ１２は分析カラム８の下流に設けられている。ＢＰＲ１０及び１２はそれぞれ、移動相供給部２によって送液される移動相溶媒中の二酸化炭素を超臨界状態にするために流路内の圧力を所定の圧力に制御するためのものである。 The BPR 10 is provided on the flow path branched from the flow path between the autosampler 6 and the analysis column 8, and the BPR 12 is provided downstream of the analysis column 8. The BPRs 10 and 12, respectively, are for controlling the pressure in the flow path to a predetermined pressure in order to bring the carbon dioxide in the mobile phase solvent sent by the mobile phase supply unit 2 into a supercritical state.

検出器１４はＢＰＲ１２のさらに下流に設けられている。検出器１４は、分析カラム８で分離した成分を検出して定量を行なうためのものであり、例えば質量分析計（ＭＳ）によって実現される。 The detector 14 is provided further downstream of the BPR 12. The detector 14 is for detecting and quantifying the components separated by the analysis column 8, and is realized by, for example, a mass spectrometer (MS).

制御部２８は、このオンラインＳＦＥ−ＳＦＣシステム１の動作制御を行なうためのものである。制御部２８は、例えば、マイクロコンピュータなどの演算素子やその演算素子によって実行されるプログラムを格納した記憶素子が搭載されたコンピュータ回路によって実現される。 The control unit 28 is for controlling the operation of the online SFE-SFC system 1. The control unit 28 is realized by, for example, a computer circuit equipped with an arithmetic element such as a microcomputer or a storage element storing a program executed by the arithmetic element.

制御部２８の機能の一部として抽出部２０及び分析部３２が設けられている。抽出部２０は、抽出容器２０に封入されたキノン類を含む試料からキノン類を含む成分を抽出するための一連の抽出動作を実行するように構成されている。一連の抽出動作とは、静的抽出と動的抽出の２つの処理を含む動作である。 An extraction unit 20 and an analysis unit 32 are provided as part of the functions of the control unit 28. The extraction unit 20 is configured to perform a series of extraction operations for extracting a component containing quinones from a sample containing quinones enclosed in an extraction container 20. The series of extraction operations is an operation including two processes of static extraction and dynamic extraction.

静的抽出とは、システム１の流路構成を図４に示されるような状態にして、抽出容器２０に移動相供給部２から所定の組成をもった第１の移動相溶媒を供給し、抽出容器２０内を第１の移動相溶媒で満たして一定時間（例えば２分）静置する処理である。この静的抽出により、試料中のキノン類を含む成分が第１の移動相溶媒に抽出される。 In the static extraction, the flow path configuration of the system 1 is set as shown in FIG. 4, and the first mobile phase solvent having a predetermined composition is supplied from the mobile phase supply unit 2 to the extraction container 20. This is a process in which the inside of the extraction container 20 is filled with the first mobile phase solvent and allowed to stand for a certain period of time (for example, 2 minutes). By this static extraction, the component containing quinones in the sample is extracted into the first mobile phase solvent.

動的抽出とは静的抽出の後で実施される処理であり、システム１の流路構成を図５に示されるような状態にして、抽出容器２０内の第１の移動相溶媒を分析カラム８に通液し、試料から第１の移動相溶媒に抽出されたキノン類を含む成分を分析カラム８に捕捉させて濃縮する処理である。 Dynamic extraction is a process performed after static extraction, and the flow path configuration of the system 1 is set as shown in FIG. 5, and the first mobile phase solvent in the extraction container 20 is analyzed. This is a process in which the liquid is passed through the sample 8 and the components containing quinones extracted from the sample into the first mobile phase solvent are captured by the analysis column 8 and concentrated.

上記一連の抽出動作において使用する第１の移動相溶媒は、静的抽出において抽出容器２０内の試料からキノン類を抽出し、かつ動的抽出において分析カラム８からキノン類を溶出させないように調整されたものである。分析カラム８がナフチルエチル基結合型のカラムである場合には、第１の移動相溶媒として、例えば、二酸化炭素とモディファイアとの比率が９：１（アセトニトリル濃度１０％）の組成をもつものを用いることができる。 The first mobile phase solvent used in the above series of extraction operations is adjusted so that quinones are extracted from the sample in the extraction container 20 in static extraction and quinones are not eluted from the analysis column 8 in dynamic extraction. It was done. When the analysis column 8 is a naphthylethyl group-bonded column, the first mobile phase solvent has a composition of, for example, a ratio of carbon dioxide to a modifier of 9: 1 (acetonitrile concentration 10%). Can be used.

上記一連の抽出動作を複数回繰り返すことにより、抽出容器２０内の試料からのキノン類の抽出効率が向上する。このため、抽出部３０は、上記一連の抽出動作を所定回数（例えば３回）繰り返すように構成されていることが好ましい。 By repeating the above series of extraction operations a plurality of times, the efficiency of extracting quinones from the sample in the extraction container 20 is improved. Therefore, it is preferable that the extraction unit 30 is configured to repeat the series of extraction operations a predetermined number of times (for example, three times).

分析部３２は、上記一連の抽出動作が終了した後で、システム１の流路構成を図６に示されるような状態にして、第１の移動相溶媒とは組成の異なる第２の移動相溶媒を分析カラム８に通液し、分析カラム８に捕捉されたキノン類を他の成分と分離しながら溶出させ、検出器１４に導くように構成されている。 After the series of extraction operations are completed, the analysis unit 32 changes the flow path configuration of the system 1 to the state shown in FIG. 6, and sets the second mobile phase having a composition different from that of the first mobile phase solvent. The solvent is passed through the analysis column 8 to elute the quinones trapped in the analysis column 8 while separating them from other components, and the solvent is guided to the detector 14.

上記分析動作において用いられる第２の移動相溶媒は、分析カラム８からのキノン類の溶出力が第１の移動相溶媒よりも高いものである。第２の移動相溶媒は、モディファイアの濃度を第１の移動相溶媒よりも高くすることによって実現される。なお、第２の移動相溶媒のモディファイア濃度を時間とともに上昇させるようにしてもよい。 The second mobile phase solvent used in the above analysis operation has a higher elution output of quinones from the analysis column 8 than the first mobile phase solvent. The second mobile phase solvent is realized by making the concentration of the modifier higher than that of the first mobile phase solvent. The modifier concentration of the second mobile phase solvent may be increased with time.

このオンラインＳＦＥ−ＳＦＣシステム１において実施されるキノン類分析方法について、図１とともに図２のフローチャートを用いて説明する。 The quinone analysis method carried out in this online SFE-SFC system 1 will be described with reference to FIG. 1 using the flowchart of FIG.

初期状態では、オンラインＳＦＥ−ＳＦＣシステム１がスタンバイ状態（図３の状態）となっている。この状態で、分析者は抽出容器２０にキノン類を含む土壌などの試料を封入し（ステップＳ１）、分析開始の指示を制御部２８に入力する。分析開始の指示が入力されると、抽出部３０はシステム１の流路構成を図４の状態にして静的抽出を実施した後（ステップＳ２）、システム１の流路構成を図５の状態にして動的抽出を実施する（ステップＳ３）。この一連の抽出動作により、試料中のキノン類を含む成分が分析カラム８に捕捉されて濃縮される。一連の抽出動作の繰返し回数が予め設定されている場合には、抽出部３０は、上記一連の抽出動作を予め設定された回数だけ繰り返す（ステップＳ４）。 In the initial state, the online SFE-SFC system 1 is in the standby state (the state shown in FIG. 3). In this state, the analyst encloses a sample such as soil containing quinones in the extraction container 20 (step S1), and inputs an instruction to start analysis to the control unit 28. When the instruction to start the analysis is input, the extraction unit 30 sets the flow path configuration of the system 1 to the state shown in FIG. 4 and performs static extraction (step S2), and then changes the flow path configuration of the system 1 to the state shown in FIG. And perform dynamic extraction (step S3). By this series of extraction operations, the components containing quinones in the sample are captured by the analysis column 8 and concentrated. When the number of repetitions of the series of extraction operations is preset, the extraction unit 30 repeats the series of extraction operations a preset number of times (step S4).

その後、分析部３２はシステム１の流路構成を図６の状態にして第２の移動相溶媒を分析カラム８に通液し、キノン類の分離分析を行なう（ステップＳ５）。検出器１４によって得られる検出信号から、試料中に含有されていたキノン類に由来するクロマトグラムが得られ、そのピーク面積を求めることによって試料中に含有されていたキノン類を定量することができる。 After that, the analysis unit 32 sets the flow path configuration of the system 1 to the state shown in FIG. 6 and passes the second mobile phase solvent through the analysis column 8 to perform separation analysis of quinones (step S5). From the detection signal obtained by the detector 14, a chromatogram derived from the quinones contained in the sample can be obtained, and the quinones contained in the sample can be quantified by determining the peak area thereof. ..

図７は、油分解微生物が保有しているキノン類として確認され、キノンプロファイリング法の主要成分であるユビキノン４種（Ｑ−８、Ｑ−９、Ｑ−１０、Ｑ−１０（Ｈ２））とメナキノン２種（Ｍ−７、Ｍ−９）についての上記キノン類分析方法による分析結果を示すクロマトグラムである。なお、（Ａ）は油分の浄化を実施する前の土壌についての分析結果であり、（Ｂ）は油分の浄化を実施して１か月が経過した後の土壌についての分析結果である。この分析では、アセトニトリル濃度が１０％である移動相（第１の移動相溶媒）を用いて静的抽出（２分）と動的抽出（２分）からなる一連の抽出動作を３回繰り返した後、移動相溶媒のアセトニトリル濃度を時間とともに上昇させて分析カラム８からキノン類を溶出させて検出器１４（ＭＳ）による検出を行なった。 FIG. 7 shows four types of ubiquinones (Q-8, Q-9, Q-10, Q-10 (H2)), which are confirmed as quinones possessed by oil-degrading microorganisms and are the main components of the quinone profiling method. It is a chromatogram which shows the analysis result by the said quinone analysis method about 2 kinds of menaquinones (M-7, M-9). In addition, (A) is the analysis result about the soil before the oil purification was carried out, and (B) is the analysis result about the soil after one month has passed after the oil purification was carried out. In this analysis, a series of extraction operations consisting of static extraction (2 minutes) and dynamic extraction (2 minutes) was repeated 3 times using a mobile phase (first mobile phase solvent) having an acetonitrile concentration of 10%. After that, the acetonitrile concentration of the mobile phase solvent was increased with time to elute the quinones from the analysis column 8, and the detection was performed by the detector 14 (MS).

図７からわかるように、上記キノン類分析方法によっていずれのキノン類もピーク波形として検出することができている。このことから、キノン類の抽出から分析までの処理を１つの分析カラム８を用いてオンラインで完結させることが可能であることが立証された。 As can be seen from FIG. 7, any quinone can be detected as a peak waveform by the above-mentioned quinone analysis method. From this, it was proved that the process from the extraction of quinones to the analysis can be completed online by using one analysis column 8.

また、土壌中に含まれるキノン類の量は土壌中の油分量に比例することがわかっている。図７の（Ａ）と（Ｂ）を比較すると、油分を浄化して１か月が経過した土壌中のキノン類の量は油分を浄化する前の土壌中のキノン類の量よりも減少しており、分析によってキノン類を正確に検出できていることがわかる。 It is also known that the amount of quinones contained in the soil is proportional to the amount of oil in the soil. Comparing (A) and (B) in FIG. 7, the amount of quinones in the soil one month after the oil was purified was smaller than the amount of quinones in the soil before the oil was purified. It can be seen that quinones can be detected accurately by analysis.

また、図８は、静的抽出（２分）と動的抽出（２分）からなる一連の抽出動作の繰返し回数によるキノン類（Ｑ−９）の抽出率の検証結果を示すクロマトグラムであり、（Ａ）は繰り返し回数が１回の場合、（Ｂ）は繰り返し回数が３回の場合である。この結果からわかるように、一連の抽出動作を１回だけ実施しても試料からのキノン類の抽出量は僅かであるが、一連の抽出動作を複数回繰り返すことによってキノン類の抽出量が大幅に上昇している。この結果から、キノンプロファイリング法の正確性に影響を与える試料からのキノン類の抽出率を大きく向上させるために、一連の抽出動作を繰り返すことが有効であることが立証された。 Further, FIG. 8 is a chromatogram showing the verification result of the extraction rate of quinones (Q-9) by the number of repetitions of a series of extraction operations consisting of static extraction (2 minutes) and dynamic extraction (2 minutes). , (A) is the case where the number of repetitions is one, and (B) is the case where the number of repetitions is three times. As can be seen from this result, the amount of quinones extracted from the sample is small even if the series of extraction operations is performed only once, but the amount of quinones extracted is large by repeating the series of extraction operations multiple times. Is rising to. From this result, it was proved that it is effective to repeat a series of extraction operations in order to greatly improve the extraction rate of quinones from the sample, which affects the accuracy of the quinone profiling method.

１ オンラインＳＦＥ−ＳＦＣシステム
２ 移動相供給部
４ ＳＦＥ部
６ オートサンプラ
８ 分析カラム
１０，１２ 背圧制御バルブ（ＢＰＲ）
１４ 検出器
１６ 二酸化炭素ポンプ
１８ モディファイアポンプ
２０ 抽出容器
２２，２４ 流路切替バルブ
２６ カラムオーブン
２８ 制御部
３０ 抽出部
３２ 分析部1 Online SFE-SFC system 2 Mobile phase supply unit 4 SFE unit 6 Autosampler 8 Analytical column 10, 12 Back pressure control valve (BPR)
14 Detector 16 Carbon dioxide pump 18 Modifier pump 20 Extraction container 22, 24 Flow path switching valve 26 Column oven 28 Control unit 30 Extraction unit 32 Analysis unit

Claims (5)

超臨界状態の二酸化炭素とモディファイアからなる移動相溶媒を供給するための移動相供給部、試料が封入され、前記移動相供給部から供給される前記移動相溶媒によって前記試料から成分を抽出するための抽出容器、前記試料から抽出された成分の分離を行なうための分析カラム、及び前記分析カラムで分離された成分を検出するための検出器を有し、前記抽出容器内で試料中に含まれる成分を前記移動相溶媒に抽出した後、前記試料から抽出された成分を含む前記移動相溶媒を前記分析カラムに導いて分離し、前記分析カラムで分離された成分を前記検出器により検出するように構成されたオンラインＳＦＥ−ＳＦＣシステムを用いたキノン類分析方法であって、
前記抽出容器にキノン類を含む試料を封入する試料封入ステップと、
前記試料封入ステップの後で、前記移動相供給部から、前記試料からキノン類を含む成分を抽出しながら前記分析カラムから前記キノン類を含む成分を溶出させないように調整された第１の移動相溶媒を前記抽出容器に供給し、前記抽出容器内が前記第１の移動相溶媒で満たされた状態で静置した後、前記試料から抽出された成分を含む前記第１の移動相溶媒を前記分析カラムに通液し、前記第１の移動相溶媒に含まれる成分のうち少なくともキノン類を含む成分を前記分析カラムに捕捉させる一連の抽出動作を複数回繰り返して実行し、前記分析カラムにおいて前記キノン類を含む成分を濃縮する抽出ステップと、
前記抽出ステップの後で、前記一連の抽出動作により前記分析カラムに捕捉された成分を前記分析カラムから溶出させるための第２の移動相溶媒を前記移動相供給部から供給して前記分析カラムに通液し、前記分析カラムにおいて濃縮された成分を分離しながら溶出させて前記検出器により検出する分析ステップと、を備え、
前記分析カラムとして、担体にナフチルエチル基を結合させた分離媒体が充填されているナフチルエチル基結合型のカラム、または担体にピレニルエチル基を結合させた分離媒体が充填されているピレニルエチル基結合型のカラムを用い、前記モディファイアとしてアセトニトリルを用いる、
キノン類分析方法。 A mobile phase supply unit and a sample for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state are enclosed, and components are extracted from the sample by the mobile phase solvent supplied from the mobile phase supply unit. It has an extraction container for the purpose, an analysis column for separating the components extracted from the sample, and a detector for detecting the components separated by the analysis column, and is contained in the sample in the extraction container. After extracting the components to be separated into the mobile phase solvent, the mobile phase solvent containing the components extracted from the sample is guided to the analysis column and separated, and the components separated by the analysis column are detected by the detector. It is a quinone analysis method using the online SFE-SFC system configured as described above.
A sample encapsulation step of encapsulating a sample containing quinones in the extraction container,
After the sample encapsulation step, the first mobile phase is adjusted so as not to elute the component containing the quinone from the analysis column while extracting the component containing the quinone from the sample from the mobile phase supply unit. The solvent is supplied to the extraction container, and the extraction container is allowed to stand in a state of being filled with the first mobile phase solvent, and then the first mobile phase solvent containing the component extracted from the sample is used. A series of extraction operations in which the liquid is passed through the analysis column and the components containing at least quinones among the components contained in the first mobile phase solvent are captured by the analysis column are repeatedly executed a plurality of times, and the above-mentioned extraction operation is performed in the analysis column. Extraction steps to concentrate components containing quinones,
After the extraction step, a second mobile phase solvent for eluting the components captured in the analysis column by the series of extraction operations from the analysis column is supplied from the mobile phase supply unit to the analysis column. It comprises an analysis step of passing a liquid, eluting while separating the concentrated components in the analysis column, and detecting by the detector .
The analytical column is a naphthylethyl group-bonded column packed with a separation medium having a naphthylethyl group bonded to a carrier, or a pyrenylethyl group-bonded column filled with a separation medium having a pyrenylethyl group bonded to a carrier. A column is used and acetonitrile is used as the modifier.
Quinone analysis method. 前記抽出ステップにおいて前記一連の抽出動作を３回以上繰り返す、請求項１に記載のキノン類分析方法。 The quinone analysis method according to claim 1, wherein the series of extraction operations is repeated three or more times in the extraction step. 前記第２の移動相溶媒の前記モディファイアの濃度を前記第１の移動相溶媒よりも高くする、請求項１又は２に記載のキノン類分析方法。 The quinone analysis method according to claim 1 or 2, wherein the concentration of the modifier of the second mobile phase solvent is higher than that of the first mobile phase solvent. 前記分析ステップにおいて、前記第２の移動相溶媒の前記モディファイアの濃度を時間とともに上昇させる、請求項３に記載のキノン類分析方法。 The quinone analysis method according to claim 3, wherein in the analysis step, the concentration of the modifier of the second mobile phase solvent is increased with time. 超臨界状態の二酸化炭素とモディファイアからなる移動相溶媒を供給するための移動相供給部、試料が封入され、前記移動相供給部から供給される前記移動相溶媒によって前記試料から成分を抽出するための抽出容器、前記試料から抽出された成分の分離を行なうための分析カラム、及び前記分析カラムで分離された成分を検出するための検出器を有し、前記抽出容器内で試料中に含まれる成分を前記移動相溶媒に抽出した後、前記試料から抽出された成分を含む前記移動相溶媒を前記分析カラムに導いて分離し、前記分析カラムで分離された成分を前記検出器により検出するように構成されたオンラインＳＦＥ−ＳＦＣシステムであって、
当該オンラインＳＦＥ−ＳＦＣシステムの動作制御を行なう制御部は、
キノン類を含む試料が封入された前記抽出容器に、前記試料からキノン類を含む成分を抽出しながら前記分析カラムから前記キノン類を含む成分を溶出させないように調整された第１の移動相溶媒を供給して前記抽出容器を前記第１の移動相溶媒で満たして静置した後、前記試料から抽出された成分を含む前記抽出容器内の前記第１の移動相溶媒を前記分析カラムに通液し、前記第１の移動相溶媒中の成分のうち少なくともキノン類を含む成分を前記分析カラムに捕捉させる一連の抽出動作を複数回繰り返して実行し、前記分析カラムにおいて前記キノン類を含む成分を濃縮するように構成された抽出部と、
前記一連の抽出動作が複数回繰り返して実行された後で、前記分析カラムにおいて濃縮された成分を前記分析カラムから溶出させるための第２の移動相溶媒を前記分析カラムに通液するように構成された分析部と、を備え、
前記分析カラムは、担体にナフチルエチル基を結合させた分離媒体が充填されているナフチルエチル基結合型のカラムまたは担体にピレニルエチル基を結合させた分離媒体が充填されているピレニルエチル基結合型のカラムであり、前記モディファイアはアセトニトリルである、オンラインＳＦＥ−ＳＦＣシステム。 A mobile phase supply unit and a sample for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state are enclosed, and components are extracted from the sample by the mobile phase solvent supplied from the mobile phase supply unit. It has an extraction container for the purpose, an analysis column for separating the components extracted from the sample, and a detector for detecting the components separated by the analysis column, and is contained in the sample in the extraction container. After extracting the components to be separated into the mobile phase solvent, the mobile phase solvent containing the components extracted from the sample is guided to the analysis column and separated, and the components separated by the analysis column are detected by the detector. An online SFE-SFC system configured to
The control unit that controls the operation of the online SFE-SFC system is
A first mobile phase solvent adjusted so as not to elute the component containing quinone from the analysis column while extracting the component containing quinone from the sample in the extraction container containing the sample containing quinone. The extraction container is filled with the first mobile phase solvent and allowed to stand, and then the first mobile phase solvent in the extraction container containing the components extracted from the sample is passed through the analysis column. A series of extraction operations in which the components in the first mobile phase solvent are liquid and contain at least quinones in the analysis column are repeatedly executed a plurality of times, and the components containing the quinones in the analysis column. With an extractor configured to concentrate
After the series of extraction operations are repeated a plurality of times, a second mobile phase solvent for eluting the concentrated components in the analysis column is passed through the analysis column. With the analysis department ,
The analysis column is a naphthylethyl group-bonded column in which a carrier is filled with a separation medium having a naphthylethyl group bonded, or a pyrenylethyl group-bonded column in which a separation medium in which a pyrenylethyl group is bonded to a carrier is packed. The online SFE-SFC system , wherein the modifier is acetonitrile.
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