WO2019163245A1

WO2019163245A1 - Quinone analysis method and online sfe-sfc system for implementing said method

Info

Publication number: WO2019163245A1
Application number: PCT/JP2018/044126
Authority: WO
Inventors: 恵子松本
Original assignee: 株式会社島津製作所
Priority date: 2018-02-26
Filing date: 2018-11-30
Publication date: 2019-08-29
Also published as: JPWO2019163245A1; JP6977863B2

Abstract

This quinone analysis method comprises: a sample sealing step for sealing a sample including quinones in an extraction container; an extraction step for, after the sample sealing step and as a series of extraction operations, carrying out "static extraction" in which a first mobile phase solvent for extracting components including quinones from the sample is supplied from a mobile phase supply unit to the extraction container and the extraction container is left still in a state of being filled with the first mobile phase solvent, and then carrying out "dynamic extraction" in which the first mobile phase solvent including components extracted from the sample is allowed to flow through the analysis column and at least the components including quinones from among the components included in the first mobile phase solvent are captured by the analysis column; and an analysis step for supplying, from the mobile phase supply unit, a second mobile phase solvent for eluting, from the analysis column, the components captured by the analysis column as a result of the series of extraction operations, causing the second mobile phase solvent to flow through the analysis column, separating and eluting the components captured by the analysis column, and detecting the same using a detector.

Description

Method for analyzing quinones and on-line SFE-SFC system for carrying out the method

The present invention relates to a method for analyzing quinones and an on-line SFE-SFC system for carrying out the method.

One of the methods for observing the activity of microorganisms in a sample such as soil is a quinone profiling method (see Patent Document 1). The quinone profiling method is a technique for grasping the type and amount of microbial communities present in a sample by extracting quinones, which are respiration-related substances of microorganisms, from the sample and measuring the amount thereof.

For the 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 composed of carbon dioxide in a supercritical state and an appropriate amount of organic solvent through the extraction container enclosing the sample. Quinones are captured by the adsorption cartridge.

After extracting the quinones from the sample, the adsorption cartridge capturing 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. The acetone solution containing the eluted quinones is passed through another silica gel column to adsorb the quinones on the silica gel column. Then, after passing the highly volatile organic solvent through a silica gel column on which quinones are adsorbed to elute and recover the quinones, the collected liquid is evaporated by an evaporator, concentrated, washed with acetone and washed with quinone. The quinones are collected and analyzed by high performance liquid chromatograph (HPLC).

WO2006 / 118167A1 WO2016 / 031008A1

As described above, in the conventional quinone profiling method, after extraction of quinones from a sample by the SFE system, it is necessary to manually perform a process such as desorption and concentration until the extracted quinones are introduced into HPLC. there were.

The object of the present invention is to make it possible to perform from quinone extraction to analysis with high efficiency without manual operation.

The method for analyzing quinones according to the present invention is to perform from extraction of quinones to separation analysis using an online SFE-SFC system without manual operation.

The online SFE-SFC system is a system configured to be able to perform on-line extraction of sample components (SFE) using a supercritical fluid and supercritical fluid chromatography analysis (SFC) of the extracted components. For example, it is disclosed in Patent Document 2. Specifically, a mobile phase supply unit for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state, a sample is sealed, and the sample is supplied by the mobile phase solvent supplied from the mobile phase supply unit An extraction container for extracting a component from the sample, an analysis column for separating the component extracted from the sample, and a detector for detecting the component separated by the analysis column. After the components contained in the sample are extracted into the mobile phase solvent in the container, the mobile phase solvent containing the components extracted from the sample is guided to the analytical column and separated, and the components separated by the analytical column Is configured to be detected by the detector.

The method for analyzing quinones of the present invention includes a sample enclosing step of enclosing a sample containing quinones in the extraction container, and extracting a component containing quinones from the sample from the mobile phase supply unit after the sample enclosing step. The first mobile phase solvent to be supplied to the extraction container, and after performing “static extraction” in which the extraction container is left in a state filled with the first mobile phase solvent, the sample The first mobile phase solvent containing the component extracted from is passed through the analytical column, and the component containing at least quinones among the components contained in the first mobile phase solvent is captured by the analytical column. An extraction step for performing "dynamic extraction" as a series of extraction operations, and a second mobile phase solvent for eluting components captured by the analysis column by the series of extraction operations from the analysis column. It is supplied from the parts was passed through the analytical column, and a, and analysis steps of detecting by the detector eluted while separating the captured components in the analytical column.

In the quinone analysis method of the present invention, it is preferable that the series of extraction operations including “static extraction” and “dynamic extraction” is repeated a plurality of times, preferably three times or more in the extraction step. By doing so, the extraction rate of quinones can be improved and the analysis accuracy can be increased.

As the second mobile phase solvent, a solvent having a higher concentration of the modifier than the first mobile phase solvent can be used.

In the analyzing step, the concentration of the modifier of the second mobile phase solvent may be increased with time.

The online SFE-SFC system according to the present invention has a function of fully automatically executing the above-described quinone analysis method. Specifically, a control unit that controls the operation of the online SFE-SFC system is configured to extract a component containing quinones from the sample into the extraction container in which the sample containing quinones is enclosed. After supplying the mobile phase solvent and filling the extraction container with the first mobile phase solvent and allowing to stand, the analysis of the first mobile phase solvent in the extraction container containing the component extracted from the sample is performed. An extraction unit configured to perform a series of extraction operations for causing the analytical column to capture a component containing at least quinones among components in the first mobile phase solvent, and passing through the column; And an analysis unit configured to pass the second mobile phase solvent for eluting the component captured by the analysis column by the extraction operation through the analysis column.

It is preferable that the extraction unit is configured to repeat the series of extraction operations a plurality of times. By doing so, the extraction rate of quinones can be improved and the analysis accuracy can be increased.

Examples of the analytical column for realizing the present invention include a naphthylethyl group or pyrenylethyl group-binding column in which a separation medium in which a naphthylethyl group or a pyrenylethyl group is bound to a carrier is packed. Acetonitrile is mentioned.

In the method for analyzing quinones of the present invention, an analyst encloses a sample containing quinones in an extraction container, and then leaves the sample in a state where the extraction container is filled with a first mobile phase solvent. Static extraction for extraction into one mobile phase solvent, and dynamic extraction for allowing the first mobile phase solvent in the extraction vessel to flow through the analysis column and capture components containing quinones in the analysis column. Since it is carried out as a series of extraction operations, and then the second mobile phase solvent is passed through the analytical column and eluted while separating the components captured by the analytical column and detected by the detector, From the extraction of quinones to the separation analysis can be completed on one SFE-SFC system.

Since the on-line SFE-SFC system of the present invention has a function of performing the above quinone analysis method in a fully automatic manner, processing from extraction of quinones to separation analysis can be performed with high efficiency.

It is a channel lineblock diagram showing roughly one example of an on-line SFE-SFC system. It is a flowchart for demonstrating the analysis method of quinones using the online SFE-SFC system of the Example. It is a figure which shows the flow-path structure of the online SFE-SFC system at the time of standby. It is a figure which shows the flow-path structure of the online SFE-SFC system at the time of static extraction. It is a figure which shows the flow-path structure of the online SFE-SFC system at the time of dynamic extraction. It is a figure which shows the flow-path structure of the online SFE-SFC system at the time of analysis. It is a chromatogram which shows an example of the analysis result by the analysis method of quinones using the online SFE-SFC system of the Example, (A) is the analysis result of the soil before purification, (B) is the soil of the soil after purification. It is an analysis result. It is a graph which shows the verification result of the extraction effect of quinones by the repetition frequency of a series of extraction operation | movement, (A) is the case where the repetition frequency is 1 time, (B) is the case where the repetition frequency is 3.

Hereinafter, a method for analyzing quinones and an online SFE-SFC system for carrying out the method according to the present invention will be described with reference to the drawings.

FIG. 1 shows a schematic configuration diagram of an embodiment of an online SFE-SFC system suitable for carrying out a quinone analysis method.

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 analytical column 8, back pressure control valves (BPR) 10, 12, a detector 14, and a control unit. 28.

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 liquid, the pressure in the flow path is controlled by the

BPR

10 or 12 so that the carbon dioxide flows in a supercritical state. Examples of the modifier include acetonitrile.

The SFE unit 4 includes an extraction container 20 and flow

path switching valves

22 and 24. The flow

path switching valves

22 and 24 are for switching the connection destination 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), static extraction (see FIG. 4), dynamic extraction (see FIG. 5) and analysis (see FIG. 3). A state for performing each operation of FIG. 6) can be taken. Details of static extraction (see FIG. 4), dynamic extraction (see FIG. 5), and analysis (see FIG. 6) will be described later.

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, for example, injecting a standard sample into a flow path when acquiring measurement data of the standard sample.

The analysis column 8 is provided further downstream of the autosampler 6. The analytical 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 accommodated in the column oven 26 and the temperature is adjusted.

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. Each of the BPRs 10 and 12 is for controlling the pressure in the flow path to a predetermined pressure in order to bring carbon dioxide in the mobile phase solvent fed by the mobile phase supply unit 2 into a supercritical state.

The detector 14 is provided further downstream of the BPR 12. The detector 14 is for detecting a component separated by the analysis column 8 and performing quantification, and is realized by, for example, a mass spectrometer (MS).

The control unit 28 is for performing operation control of the online SFE-SFC system 1. The control unit 28 is realized by, for example, a computer circuit on which an arithmetic element such as a microcomputer and a storage element storing a program executed by the arithmetic element are mounted.

The extraction unit 20 and the analysis unit 32 are provided as part of the function 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 the extraction container 20. A series of extraction operations are operations including two processes of static extraction and dynamic extraction.

With static extraction, the flow path configuration of the system 1 is set as shown in FIG. 4, and a first mobile phase solvent having a predetermined composition is supplied to the extraction container 20 from the mobile phase supply unit 2. In this process, the inside of the extraction container 20 is filled with the first mobile phase solvent and allowed to stand for a certain time (for example, 2 minutes). By this static extraction, a component containing quinones in the sample is extracted into the first mobile phase solvent.

The 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 with an analysis column. 8 is a process in which a component containing quinones extracted from the sample into the first mobile phase solvent is captured by the analytical column 8 and concentrated.

The first mobile phase solvent used in the above-described series of extraction operations is adjusted so that quinones are extracted from the sample in the extraction container 20 in the static extraction and the quinones are not eluted from the analytical column 8 in the dynamic extraction. It has been done. When the analytical column 8 is a naphthylethyl group-bonded column, the first mobile phase solvent has, for example, a composition in which the ratio of carbon dioxide to modifier is 9: 1 (acetonitrile concentration 10%) Can be used.

The extraction efficiency of quinones from the sample in the extraction container 20 is improved by repeating the above series of extraction operations a plurality of times. For this reason, it is preferable that the extraction unit 30 is configured to repeat the above-described series of extraction operations a predetermined number of times (for example, three times).

After the series of extraction operations is completed, the analysis unit 32 sets the flow path configuration of the system 1 as 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, and the quinones captured by the analysis column 8 are eluted while being separated from other components and guided to the detector 14.

The second mobile phase solvent used in the above analysis operation has a higher elution power 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. In addition, you may make it raise the modifier concentration of a 2nd mobile phase solvent with time.

The quinone analysis method performed in the online SFE-SFC system 1 will be described with reference to the flowchart of FIG. 2 together with FIG.

In the initial state, the online SFE-SFC system 1 is in a 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 analysis start instruction to the control unit 28. When an instruction to start analysis is input, the extraction unit 30 performs static extraction with the flow path configuration of the system 1 in the state of FIG. 4 (step S2), and then changes the flow path configuration of the system 1 to the state of FIG. Thus, dynamic extraction is performed (step S3). By this series of extraction operations, components containing quinones in the sample are captured by the analysis column 8 and concentrated. If 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).

Thereafter, 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 quinones contained in the sample is obtained, and the quinones contained in the sample can be quantified by obtaining the peak area. .

FIG. 7 shows four ubiquinone species (Q-8, Q-9, Q-10, Q-10 (H2)) that 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 types of menaquinone (M-7, M-9). In addition, (A) is an analysis result about the soil before implementing oil purification, (B) is an analysis result about the soil after implementing oil purification for one month. In this analysis, a series of extraction operations consisting of static extraction (2 minutes) and dynamic extraction (2 minutes) was repeated three times using a mobile phase (first mobile phase solvent) having an acetonitrile concentration of 10%. Thereafter, the acetonitrile concentration of the mobile phase solvent was increased with time to elute quinones from the analytical column 8, and detection was performed by the detector 14 (MS).

As can be seen from FIG. 7, any quinones can be detected as a peak waveform by the above quinone analysis method. From this, it was proved that the processing from the extraction of quinones to the analysis can be completed online by using one analytical column 8.

Also, it is known that the amount of quinones contained in the soil is proportional to the amount of oil in the soil. When (A) and (B) in FIG. 7 are compared, the amount of quinones in the soil after one month has passed since the oil was purified is less than the amount of quinones in the soil before the oil was purified. The analysis shows that quinones can be detected accurately.

FIG. 8 is a chromatogram showing the verification results 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) shows the case where the number of repetitions is 1, and (B) shows the case where the number of repetitions is three. 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 extraction amount of quinones is greatly increased by repeating the series of extraction operations multiple times. Is rising. 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 a sample that affects the accuracy of the quinone profiling method.

1 Online SFE-SFC system 2 Mobile phase supply unit 4 SFE unit 6 Autosampler 8

Analysis column

10, 12 Back pressure control valve (BPR)
DESCRIPTION OF SYMBOLS 14 Detector 16 Carbon dioxide pump 18 Modifier pump 20

Extraction container

22, 24 Flow path switching valve 26 Column oven 28 Control part 30 Extraction part 32 Analysis part

Claims

A mobile phase supply unit for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state, a sample is enclosed, and components are extracted from the sample by the mobile phase solvent supplied from the mobile phase supply unit An extraction container, an analysis column for separating components extracted from the sample, and a detector for detecting the components separated by the analysis column, and contained in the sample in the extraction container The components extracted from the sample are extracted into the mobile phase solvent, the mobile phase solvent containing the components extracted from the sample is guided to the analytical column and separated, and the components separated by the analytical column are detected by the detector. A method for analyzing quinones using an on-line SFE-SFC system configured as follows:
A sample enclosing step of enclosing a sample containing quinones in the extraction container;
After the sample sealing step, a first mobile phase solvent for extracting a component containing quinones from the sample from the mobile phase supply unit is supplied to the extraction container, and the inside of the extraction container is the first After standing in a state filled with the mobile phase solvent, the first mobile phase solvent containing the component extracted from the sample is passed through the analytical column, and the components contained in the first mobile phase solvent An extraction step for performing a series of extraction operations for causing the analytical column to capture a component containing at least quinones among
A second mobile phase solvent for eluting the component captured by the analytical column by the series of extraction operations from the analytical column is supplied from the mobile phase supply unit and passed through the analytical column, and the analytical column A quinone analysis method, comprising: an elution step of separating the components captured in the sample while separating them and detecting them by the detector.
The quinone analysis method according to claim 1, wherein the series of extraction operations are repeated a plurality of times in the extraction step.
The quinone analysis method according to claim 2, wherein the series of extraction operations is repeated three or more times in the extraction step.
The method for analyzing quinones according to any one of claims 1 to 3, wherein the concentration of the modifier of the second mobile phase solvent is higher than that of the first mobile phase solvent.
The quinone analysis method according to claim 4, wherein, in the analysis step, the concentration of the modifier of the second mobile phase solvent is increased with time.
A naphthylethyl group-binding column in which a separation medium in which a naphthylethyl group or pyrenylethyl group is bound to a carrier is packed as the analytical column is used, and acetonitrile is used as the modifier. The method for analyzing quinones according to one item.
A mobile phase supply unit for supplying a mobile phase solvent composed of carbon dioxide and a modifier in a supercritical state, a sample is enclosed, and components are extracted from the sample by the mobile phase solvent supplied from the mobile phase supply unit An extraction container, an analysis column for separating components extracted from the sample, and a detector for detecting the components separated by the analysis column, and contained in the sample in the extraction container The components extracted from the sample are extracted into the mobile phase solvent, the mobile phase solvent containing the components extracted from the sample is guided to the analytical column and separated, and the components separated by the analytical column are detected by the detector. An online SFE-SFC system configured as follows:
The controller that controls the operation of the online SFE-SFC system
A first mobile phase solvent for extracting a component containing quinones from the sample is supplied to the extraction container in which the sample containing quinones is enclosed, and the extraction container is filled with the first mobile phase solvent. The first mobile phase solvent in the extraction container containing the component extracted from the sample is passed through the analytical column, and at least quinone of the components in the first mobile phase solvent. An extraction unit configured to perform a series of extraction operations for causing the analytical column to capture a component containing a species,
An on-line SFE-SFC system comprising: an analysis unit configured to pass the second mobile phase solvent for eluting the component captured by the analysis column from the analysis column. .
The online SFE-SFC system according to claim 7, wherein the extraction unit is configured to repeat the series of extraction operations a plurality of times.
The on-line SFE-SFC according to claim 7 or 8, wherein the analytical column is a naphthylethyl group-binding column packed with a separation medium in which a naphthylethyl group is bound to a carrier, and the modifier is acetonitrile. system.