WO2003038425A1 - Liquid chromatographic mass spectrograph - Google Patents
Liquid chromatographic mass spectrograph Download PDFInfo
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- WO2003038425A1 WO2003038425A1 PCT/JP2002/011285 JP0211285W WO03038425A1 WO 2003038425 A1 WO2003038425 A1 WO 2003038425A1 JP 0211285 W JP0211285 W JP 0211285W WO 03038425 A1 WO03038425 A1 WO 03038425A1
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- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
Definitions
- the present invention relates to a liquid chromatograph mass spectrometer (LCZMS) in which a liquid chromatograph (hereinafter referred to as LC) and a mass spectrometer (hereinafter referred to as MS) are combined. It relates to a device for measuring.
- LCZMS liquid chromatograph mass spectrometer
- MALDI-TOF-MS MALDI-TOF-MS
- MALDI-TOF-MS ionization was carried out under a high vacuum, so it was necessary to re-prepare samples separated by LC for ionization. In other words, it was not possible to directly guide the sample from LC to MALD I-TOR-MS, and the analysis efficiency using MALD I-T ⁇ F-MS was very poor. Therefore, it was impossible to fractionate the target oligonucleic acid based on the information of the mass spectrum obtained by MALDI-TOF-MS.
- MS that can directly introduce a sample from LC and perform mass spectrometry includes a quadrupole mass spectrometer (Q-MS) equipped with an atmospheric pressure ion source and an ion trap mass spectrometer (IT-MS).
- Q-MS quadrupole mass spectrometer
- IT-MS ion trap mass spectrometer
- the upper limit of the measurable mass number of a Q-MS or an IT-MS equipped with an atmospheric pressure ion source is about several thousand, so that the oligonucleic acid with a large mass number exceeds the upper limit. Therefore, in order to measure oligonucleic acid with Q_MS or IT-MS, multivalent ions must be used.
- An object of the present invention is to provide an apparatus for performing analysis by LC / MS in the analysis of oligonucleic acid.
- a characteristic configuration of the present invention is a pump that sends an eluent containing an ion pair agent or a salt, a sample injector that injects a sample to be measured into a flow channel, a column that separates the introduced solution for each component, and the column.
- a liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and eluting a solution eluted from a solution with an atmospheric pressure ion source, wherein a weak base solution is merged between the column and the mass spectrometer. That is, there is provided a merging means.
- the MS / MS in which the eluate from the LC is introduced into the MS online, even if an ion pair agent necessary for the analysis of oligonucleic acid is mixed in the eluate, the MS / MS can be transferred to the MS with high sensitivity. It will be possible to analyze it.
- FIG. 1 is a schematic configuration diagram of the first embodiment.
- FIG. 2 is the total ion chromatogram (TIC) obtained by MS9.
- FIG. 3 is a mass spectrum at the retention time of each base sample peak in FIG.
- FIG. 4 is a mass spectrum of an oligo nucleotide nucleic acid sample of 40 bases.
- FIG. 5 is a schematic configuration diagram of the second embodiment.
- FIG. 6 is a schematic diagram showing the structure of the splitter 11.
- FIG. 7 is a schematic configuration diagram of a modified example of the second embodiment.
- FIG. 8 is a schematic configuration diagram of the third embodiment.
- FIG. 9 is a schematic configuration diagram of a modification of the third embodiment.
- FIG. 10 is a schematic configuration diagram of a modified example of the third embodiment. BEST MODE FOR CARRYING OUT THE INVENTION
- FIG. 1 shows a schematic configuration diagram of the first embodiment.
- the LC / MS of the present embodiment has a function of selecting a plurality of solutions (organic solvents A and B including a buffer) 1 and '2, and changing the composition of the solution while mixing them over time.
- a pump 4 with a so-called gradient elution) a sample injector 6 for introducing an oligonucleic acid sample into the flow path, a column 7 for separation of each component, and a weak base solution (eg imidazole solution) 3
- It consists of a pump 5 for joining, a mixer (coil or column) 8 for stirring and mixing the combined solution, an MS 9 for ionizing and detecting the eluate introduced from the LC, and a controller 10.
- the MS 9 used in the present invention uses an atmospheric pressure ion source such as ESI, SSI or IS as the ion source.
- an ion trap type, a time-of-flight type, and a quadrupole type device can be used for the mass spectrometer.
- the oligonucleic acid sample injected into the channel from the sample injector 6 is separated into a single component by the column 7, and then merges with the weak base solution 3 sent by the pump 5.
- the combined solution is stirred and mixed by the mixer 8 and introduced into the MS 9 so that mass information of the oligonucleic acid can be obtained.
- the weak base solution 3 used in the above method is effective even if a base having a pKb of 5.0 or more, such as a piperidine 'solution, is used in addition to the imidazole solution.
- FIG. 2 shows the total ion chromatogram (TIC) obtained by MS9.
- the TIC in FIG. 2 (a) is a result when the flow rate of the pump 5 is set to 0.0 mL Zmin and the weak base solution 3 is not added, that is, an analysis result under the same conditions as the conventional method.
- the TIC in FIG. 2 (b) is the result obtained by setting the flow rate of the pump 5 to 1.0 mLZmin and adding the weak base solution 3.
- the analysis conditions in Fig. 2 are as follows: Solution 1 contains 0.1 mol / L triethylamine acetate; 10% aqueous acetonitril solution (V / V :); Solution 2 contains 0.1 mol / L triethylamine acetate.
- oligonucleic acid sample is a mixed aqueous solution of three samples with the number of bases of 20, 30, and 40.
- the concentrations of the 20 base sample are 24 mol / L and the 30 base sample are 40 mol / L.
- the L, 40 base sample was 29 ⁇ mol ZL.
- 30 ⁇ L of the oligonucleic acid sample was injected from the sample injector 6.
- the mass number (m) of the 20 base sample is 6096
- that of the 30 base sample is 9200
- that of the 40 base sample is 123.60.
- TIC in Fig. 2 (a) only the peak of the 20-base sample could be detected.
- the 30-base sample and the 40-base sample are buried in noise components. No, it cannot be specified on TI c.
- FIG. 3 shows the mass spectrum at the retention time of each base sample peak detected in FIGS. 2 (a) and (b).
- the addition of the weak base solution 3 also increases the valency of the generated ions, and the 20-base sample is a pentavalent ion, m-noz1218, and the 30-base sample is a hexavalent ion.
- the ions of m / z 1532 and 7-valent mZz1313 were detected, and could be detected on the lower quality number side.
- the mass spectrum of the 40-base sample which was not detected at all when the weak base solution 3 was not added, was also reduced from the 7-valent mZzl 764 to the 10-valent as shown in Fig. 4. It was possible to detect ions up to m / z 1 235 with high sensitivity.
- FIG. 5 shows a second embodiment.
- an oligonucleic acid fractionation system in which a fraction collector 12 is connected via a splitter 11 for splitting an eluate from a column 7 to the configuration of the first embodiment is described.
- Fraction collector 1 2 also pump, sample injection It can be controlled by the controller 10 together with the rectifier 6, pump 5, and MS'9.
- the splitter 11 has the structure shown in FIG. 6, and has two resistance coils having different lengths inside. Due to the difference in the resistance of this coil, a certain amount of the eluate from column 7 to 1/10 to 1/1000 flows out to the MS 9 side and merges with the weak base solution 3 sent from the pump 5. Let it. In this way, the weak base solution 3 can be combined only with the flow path to the MS 9 c. The combined solution is stirred by the mixer 8 and introduced into the MS 9.
- the mass number of the oligonucleic acid and the mass number of the multiply charged ions to be collected are input in advance.
- the controller 10 sends a signal to the fraction collector 12 through a signal line.
- the fraction collector 12 separates the desired oligo nucleic acid sample into a container such as a test tube.
- the fraction collector 12 can sample the sample based on the peak signal detected by the UV detector. In this case, since the MS is performing analysis simultaneously with the fractionation, the fractionated sample can be identified by its mass number information.
- a hexagonal pulp 13 that alternates between a solid flow path and a dotted flow path at regular time intervals is connected between the column 7 and the MS 9 and the fraction collector 12.
- the eluate from column 7 is When the hexagonal pulp 13 is switched, it flows out alternately to the MS 9 and the fraction collector 12.
- the flow path of the hexagonal valve 13 should be switched, for example, every 1 second. .
- the controller 10 when the MS 9 detects a mouth massogram peak corresponding to an ion having the mass number of the target oligonucleic acid sample, the controller 10 returns to the controller 10. A signal is sent to the fraction collector 12, which collects the target oligonucleic acid sample.
- FIG. 8 shows a third embodiment.
- This embodiment is an example in which a column is added to the configuration of the first embodiment so that two types of oligonucleic acid samples can be simultaneously analyzed.
- a splitter 17 for equally dividing weak base solution 3 into two flow paths from columns 7 and 15 and a flow path for columns 7 and 15
- a 10-way valve 18 for switching the two flow paths from the outlet to one MS, a transfer solution 19 for sending the eluate reaching the 10-way pulp 18 to the MS 9, and Liquid sending pump 20 is added.
- the 10-way pulp 18 and the pump 20 can also be controlled by the same controller 10 as the pumps 4 and 14, the sample injector 6, the pump 5, and the MS9.
- the sample injector 6 can inject a sample into any of the flow paths.
- the two types of oligonucleic acid samples injected into the two channels from the sample injector 6 are separated into single components by the respective columns 7 and 15.
- the weak base solution 3 sent by the pump 5 is divided into two equal volumes by the splitter 17.
- Each of the divided weak base solutions 3 is combined with the eluate from the columns 7 and 15, respectively, and stirred by the mixers 8 and 16 to be sent to the 10-way pulp 18.
- the 10-way pulp 18 switches between the solid flow path and the dotted flow path at regular time intervals (for example, 1 second). be introduced.
- the MS 9 determines, based on the switching time interval of the valve 18, whether the detected signal is due to one of the two flow paths, and processes the two types of signals separately. As a result, mass information of the oligonucleic acid sample dissolved from the two columns 7 and 15 can be obtained by one MS 9.
- the transfer solution 19 has a role of sending the eluate from the two flow paths and a role of preventing the mixing of the two eluate in the MS ion source. Therefore, it is preferable that the transfer solution 19 does not affect the analysis in the MS 9, and the use of the weak base solution 3 already added to the eluate may increase the sensitivity of the MS 9 Can do the best.
- the weak base solution 3 methanol or the like can be used.
- the splitters 11 and 21 and the fraction collectors 12 and 22 that can be controlled by one controller 10 are connected, and the oligonucleic acid sample collection according to the number of flow paths is performed.
- System '. Use the splitters 11 and 21 shown in Fig. 6.
- FIG. 10 is a schematic configuration diagram when the amount of the oligonucleic acid sample is small. Without using splitters 11 and 21, weak base solution 3 was combined with all of the eluate from columns 7 and 15 and eluted from columns 7 and 15 by 10-way valve 18. Samples other than those to be introduced into MS 9 A nucleic acid sample is collected in its entirety.
- the action of a weak base solution facilitates the generation of multiply charged ions, so measurement with an ion trap type or quadrupole type mass spectrometer, which has a lower upper limit of the measurable mass number than T ⁇ F-MS Becomes possible.
- the efficiency of the analysis using TOF-MS can be greatly increased.
- the oligonucleic acid is fractionated using the mass number of the target oligonucleic acid and the mass number of the polyvalent ion as a signal. Becomes possible. At this time, since the fractionation is performed while taking the mass spectrum, it is possible to record the mass spectrum of the fractionated oligonucleic acid sample simultaneously with the separation as quality data of the sample.
- the liquid is transferred from the switched pulp to the MS using an independent pump, so that mixing of the samples in the ion source can be prevented.
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Abstract
It is intended to provide an apparatus for LC/MS analysis to be used in analyzing oligonucleic acids. A liquid chromatographic mass spectrograph which has a pump for feeding an eluent containing an ion pairing agent and a salt, a sample injector for injecting a sample to be analyzed into a channel, a column for fractionating the injected solution depending on components, and a mass spectrograph for ionizing the solution eluted from the column by an atmospheric ion source and detecting the same, characterized by further having a recombining means of adding a weakly basic solution between the column and the mass spectrogram. Owing to the above constitution, an oligonucleic acid can be analyzed at a high efficiency and a high sensitivity.
Description
明 細 書 Specification
液体ク口マトグラフ質量分析装置 技術分野 Liquid mouth mass spectrometer Technical field
本発明は,、液体クロマトグラフ (以下、 L C) と質量分析装置 (以下、 MS ) が結合された液体クロマ トグラフ質量分析装置 (L CZMS) に 係り、特に、オリ ゴ核酸を MSで高感度に測定するための装置に関する。 背景技術 The present invention relates to a liquid chromatograph mass spectrometer (LCZMS) in which a liquid chromatograph (hereinafter referred to as LC) and a mass spectrometer (hereinafter referred to as MS) are combined. It relates to a device for measuring. Background art
合成したオリ ゴ核酸を精製するためには、 現在は、 L Cを用いて目的 とするオリ ゴ核酸と夾雑物を分離する方法が一般的である。 この際、 逆 相、 またはイオン交換カラムで分離させるためには、 アナリティカル · ケ ミ ス ト リ ー, 1 9 9 7年 6 9卷の 1 3 2 0頁〜 1 3 2 5頁(Anal. Chem. , 1997, 69, 1320 - 1325) で論じられているように、 .適量のイオン ペア剤 (例えば、 酢酸トリェチルアンモニゥム (Τ Ε ΑΑ), 酢酸トリブ チルアンモニゥム(T B A Α), 酢酸ジプチルアンモユウム (D B AA) 等の塩) を溶離液に添加する必要がある。 At present, in order to purify the synthesized oligonucleic acid, a method of separating the target oligonucleic acid from contaminants using LC is generally used. At this time, in order to separate on a reversed phase or an ion exchange column, Analytical Chemistry, pp. 1320 to 1325, Vol. Chem., 1997, 69, 1320-1325). Appropriate amounts of ion-pairing agents (eg, triethylammonium acetate (Τ-Ε), tributylammonium acetate (TBAΑ), dibutyl acetate) It is necessary to add luanmoyuum (salts such as DB AA) to the eluent.
また、 精製したオリ ゴ核酸のマススペク トルを得るには、 オリ ゴ核酸 の質量数は数千から数万と大きいために、 質量数数万程度まで測定可能 なマ ト リ ックス支援レーザー脱離イオン化飛行時間型質量分析装置 In order to obtain a mass spectrum of the purified oligonucleic acid, since the mass number of the oligonucleic acid is as large as several thousands to tens of thousands, matrix-assisted laser desorption ionization capable of measuring up to several tens of thousands of masses Time-of-flight mass spectrometer
(M A LD I - T O F -M S ) で測定していた。 しかしながら、 MALDI — T O F— MSを用いた場合、 イオン化が高真空化で行われるため、 L Cで分離した試料をイオン化用に再調製する作業が必要であった。 即 ち、 L Cからの試料を直接 M A LD I — TO R— MSに導く ことは不可 能であり、 MALD I 一 T〇 F— MSを用いた分析能率は大変悪かった。
従って、 MA LD I —TO F— MSで得たマススぺク トルの情報に基づ いて目的とするオリ ゴ核酸を分取する事も不可能であった。 (MA LD I-TOF-MS). However, when MALDI-TOF-MS was used, ionization was carried out under a high vacuum, so it was necessary to re-prepare samples separated by LC for ionization. In other words, it was not possible to directly guide the sample from LC to MALD I-TOR-MS, and the analysis efficiency using MALD I-T〇F-MS was very poor. Therefore, it was impossible to fractionate the target oligonucleic acid based on the information of the mass spectrum obtained by MALDI-TOF-MS.
一方、 L Cからの試料を直接導入して質量分析できる MSとしては、 大気圧イオン源を備えた四重極型質量分析装置 (Q— MS) やイオント ラップ型質量分析装置 ( I T—MS) が知られている。 そこで、 L Cか らの試料がオンラインで M Sに導かれた装置、 即ち L CZM Sでオリ ゴ 核酸の分離と質量分析を行おう とした場合、 上記に示すような大気圧ィ オン源を備えた MSを用いる必要がある。 また、 大気圧イオン源と して は、 エレク トロスプレイイオン化法 (E S I ), ソニックスプレイイオン 化法 (S S I ) 又はイオンスプレイ ( I S) 等が一般的に用いられる。 発明の開示 On the other hand, MS that can directly introduce a sample from LC and perform mass spectrometry includes a quadrupole mass spectrometer (Q-MS) equipped with an atmospheric pressure ion source and an ion trap mass spectrometer (IT-MS). Are known. Therefore, when the separation of oligonucleic acids and mass spectrometry were to be performed using a device in which the sample from the LC was led to the MS online, that is, LCZMS, the atmospheric pressure ion source as shown above was provided. It is necessary to use MS. As an atmospheric pressure ion source, an electrospray ionization method (ESI), a sonic spray ionization method (SSI), an ion spray (IS), or the like is generally used. Disclosure of the invention
しかしながら大気圧イオン源を備えた Q— MSや I T一 MSは、 測定 可能な質量数上限が数千程度であるため、 質量数の大きなオリ ゴ核酸は 上限を超えてしま う。 そのため、 オリ ゴ核酸を Q _M Sや I T— M Sで 測定するには、 多価ィオンを用いなければならない。 However, the upper limit of the measurable mass number of a Q-MS or an IT-MS equipped with an atmospheric pressure ion source is about several thousand, so that the oligonucleic acid with a large mass number exceeds the upper limit. Therefore, in order to measure oligonucleic acid with Q_MS or IT-MS, multivalent ions must be used.
また、 オリ ゴ核酸の L Cでの分離に必要なイオンペア剤が MSに導入 される試料に混入していると、 E S I , S S I又は I S等の大気圧ィォ ン源でのイオンの生成が妨害され(イオンサブレッショ ン)、 著しい MS の感度低下を招く。 In addition, if the ion-pairing agent necessary for the separation of oligonucleic acid by LC is mixed into the sample to be introduced into the MS, the generation of ions by an atmospheric pressure ion source such as ESI, SSI, or IS is hindered. (Ion cancellation), causing a significant decrease in MS sensitivity.
このような状況を防ぐための方法と して、 例えば、 RAPID To prevent this situation, for example, RAPID
COMMUNICATIONS IN MASS SPECTR0MET0RY, VOL.9, 97-102 (1995)に開 示されているように、 MSに導入される試料にィミダゾール溶液等の弱 塩基の溶液を混ぜ、 E S I -MSで測定することが提案されている。 As disclosed in COMMUNICATIONS IN MASS SPECTRMETRY, VOL. 9, 97-102 (1995), it is possible to mix a sample to be introduced into MS with a solution of a weak base such as imidazole solution and measure it with ESI-MS. Proposed.
しかしながら、 上記文献には、 L Cと MSをオンラインで接続した装
置において、 MSに導入される試料に弱塩基溶液を効率よく混合させる ための構成は何等示されていない。 However, in the above document, the LC and MS were connected online. However, there is no description of a configuration for efficiently mixing a weak base solution with a sample to be introduced into the MS in the apparatus.
従って、 L CZMSでオリ ゴ核酸を分離, 精製し、 かつ感度良くマス スぺク トルを検出することは従来では行われておらず、 ォリ ゴ核酸の分 離と質量分析をオンラインで行う事は不可能であった。 Therefore, separation and purification of oligonucleic acid by LCZMS and detection of a mass spectrum with high sensitivity have not been conventionally performed, and it is necessary to perform online separation of oligonucleic acid and mass spectrometry. Was impossible.
本発明は、 オリ ゴ核酸の分析において、 L C/M Sで分析を行うため の装置を提供することを目的と したものである。 An object of the present invention is to provide an apparatus for performing analysis by LC / MS in the analysis of oligonucleic acid.
本発明の特徴的な構成は、 イオンペア剤や塩を含む溶離液を送液する ポンプ、 測定対象試料を流路内に注入するサンプルインジェクタ、 導入 された溶液を成分ごとに分離するカラム、 当該カラムから溶出してきた 溶液を大気圧イオン源によってイオン化し検出する質量分析装置を有す る液体クロマトグラフ質量分析装置であって、 前記カラムと前記質量分 析装置の間に、弱塩基の溶液を合流させる合流手段を備えたことである。 本発明においては、 L Cからの溶出液をオンラインで MSに導入する L C /MSにおいて、 オリ ゴ核酸の分析時に必要なイオンペア剤が溶離 液に混入されている場合であっても、 感度良く MSにて分析することか 可能になるものである。 A characteristic configuration of the present invention is a pump that sends an eluent containing an ion pair agent or a salt, a sample injector that injects a sample to be measured into a flow channel, a column that separates the introduced solution for each component, and the column. A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and eluting a solution eluted from a solution with an atmospheric pressure ion source, wherein a weak base solution is merged between the column and the mass spectrometer. That is, there is provided a merging means. In the present invention, in the LC / MS in which the eluate from the LC is introduced into the MS online, even if an ion pair agent necessary for the analysis of oligonucleic acid is mixed in the eluate, the MS / MS can be transferred to the MS with high sensitivity. It will be possible to analyze it.
図面の簡単な説明 BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 第 1の実施例の概略構成図である。 FIG. 1 is a schematic configuration diagram of the first embodiment.
第 2図は、 MS 9で得られた全イオンクロマ トグラム (T I C) であ る。 FIG. 2 is the total ion chromatogram (TIC) obtained by MS9.
第 3図は、 第 2図の各塩基試料ピークの保持時間におけるマススぺク トルである。 FIG. 3 is a mass spectrum at the retention time of each base sample peak in FIG.
第 4図は、 4 0塩基のオリ ゴ核酸試料のマススぺク トルである。 FIG. 4 is a mass spectrum of an oligo nucleotide nucleic acid sample of 40 bases.
第 5図は、 第 2の実施例の概略構成図である。
第 6図は、 スプリ ッタ 1 1の構造を示した模式図である。 FIG. 5 is a schematic configuration diagram of the second embodiment. FIG. 6 is a schematic diagram showing the structure of the splitter 11.
第 7図は、 第 2の実施例の変形例の概略構成図である。 FIG. 7 is a schematic configuration diagram of a modified example of the second embodiment.
第 8図は、 第 3の実施例の概略構成図である。 FIG. 8 is a schematic configuration diagram of the third embodiment.
第 9図は、 第 3の実施例の変形例の概略構成図である。 FIG. 9 is a schematic configuration diagram of a modification of the third embodiment.
第 1 0図は、 第 3の実施例の変形例の概略構成図である。 発明を実施するための最良の形態 FIG. 10 is a schematic configuration diagram of a modified example of the third embodiment. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施例を説明する。 Hereinafter, examples of the present invention will be described.
(実施例 1 ) (Example 1)
第 1図に、 第 1の実施例の概略構成図を示す。 本実施例の L C / M S は、 複数の溶液 (バッファを含む有機溶媒 A , B ) 1 , ' 2を選択し、 時 間と共にそれらを混合しながら溶液の組成を変えて送液できる機能 (い わゆるグラジェント溶出) を持ったポンプ 4, オリ ゴ核酸試料を流路内 に導入するサンプルインジヱクタ 6, 成分毎の分離を行うカラム 7 , 更 に弱塩基溶液 (例えばイミダゾール溶液) 3を合流させるためのポンプ 5 , 合流した溶液を攪拌, 混合させるミキサ (コイルまたはカラム) 8, L Cから導入された溶出液をイオン化し検知する M S 9 , コントローラ 1 0から構成されている。 本発明で用いられる M S 9は、 イオン源に E S I, S S Iや I S等の大気圧イオン源を用いる。 また、 質量分析部 には、 イオントラップ型, 飛行時間型, 四重極型の各装置を用いること ができる。 FIG. 1 shows a schematic configuration diagram of the first embodiment. The LC / MS of the present embodiment has a function of selecting a plurality of solutions (organic solvents A and B including a buffer) 1 and '2, and changing the composition of the solution while mixing them over time. A pump 4 with a so-called gradient elution), a sample injector 6 for introducing an oligonucleic acid sample into the flow path, a column 7 for separation of each component, and a weak base solution (eg imidazole solution) 3 It consists of a pump 5 for joining, a mixer (coil or column) 8 for stirring and mixing the combined solution, an MS 9 for ionizing and detecting the eluate introduced from the LC, and a controller 10. The MS 9 used in the present invention uses an atmospheric pressure ion source such as ESI, SSI or IS as the ion source. In addition, an ion trap type, a time-of-flight type, and a quadrupole type device can be used for the mass spectrometer.
サンプルィンジヱクタ 6から流路に注入されたォリ ゴ核酸試料は、 力 ラム 7によって単一成分に分離された後、 ポンプ 5により送られる弱塩 基溶液 3 と合流する。合流した溶液はミキサ 8によつて攪拌,混合され、 M S 9に導入され、 ォリ ゴ核酸の質量情報を得ることができる。 本発明
で用いられる弱塩基溶液 3は、 ィミダゾール溶液以外にピペリジン'溶液 等、 p K b 5. 0以上の塩基を用いても効果がある。 The oligonucleic acid sample injected into the channel from the sample injector 6 is separated into a single component by the column 7, and then merges with the weak base solution 3 sent by the pump 5. The combined solution is stirred and mixed by the mixer 8 and introduced into the MS 9 so that mass information of the oligonucleic acid can be obtained. The present invention The weak base solution 3 used in the above method is effective even if a base having a pKb of 5.0 or more, such as a piperidine 'solution, is used in addition to the imidazole solution.
また、 上記のポンプ 4, サンプルインジヱクタ 6 , ポンプ 5, M S 9 はすべてコントローラ 1 0で制御することが可能である。 Further, the above pump 4, sample injector 6, pump 5, and MS 9 can all be controlled by the controller 10.
第 2図は、 M S 9によって得られる全イオンクロマ トグラム (T I C) の である。第 2図 ( a ) の T I Cは、 ポンプ 5の流量を 0. O m L Zmin として弱塩基溶液 3を加えない場合の結果であり、 即ち、 従来の方法と 同様の条件の分析結果である。 第 2図 (b ) の T I Cは、 ポンプ 5の流 量を 1. 0 m LZmin として弱塩基溶液 3を加えて得られた結果である。 第 2図の分析条件は、 溶液 1は 0. 1 mol/ L酢酸ト リェチルァミン含 有 1 0 %ァセ トニ トリル水溶液( V / V:)、溶液 2は 0. 1 mol/ L酢酸ト リェチルァミン含有 2 0 %ァセ トニト リル水溶液 ( V / V ) で、 分離力 ラム 7にカラムサイズ 4. 6 X 1 5 0 mm ( 5 μ m充填材) の 0 D S (オタ タデシルシラン) カラム、 弱塩基溶液 3に 0. 1 molZ Lイミダゾ一ルの ァセ トニトリル溶液を用いた。 ポンプ 4の流量は 0. 5 m L /minで、 溶 液 1 と 2の組成を、 溶液 1 溶液 2 = 1 0 0Z 0から始めて 2 0分かけ て溶液 1 Ζ溶液 2 = 5 0Ζ 5 0まで変化させる直線グラジェント溶出を 行った。 オリ ゴ核酸試料は、 塩基数が 2 0, 3 0, 4 0の 3試料の混合 水溶液で、 それぞれの濃度を 2 0塩基試料が 2 4 molノ L、 3 0塩基試 料が 4 0 mol/ L、 4 0塩基試料が 2 9 μ molZ Lと した。 このオリ ゴ 核酸試料 3 0 μ Lをサンプルィンジヱクタ 6より注入した。 ちなみに、 2 0塩基試料の質量数 (m) は 6 0 9 6、 3 0塩基試料は 9 2 0 0、 4 0塩基試料は 1 2 3 6 0である。 FIG. 2 shows the total ion chromatogram (TIC) obtained by MS9. The TIC in FIG. 2 (a) is a result when the flow rate of the pump 5 is set to 0.0 mL Zmin and the weak base solution 3 is not added, that is, an analysis result under the same conditions as the conventional method. The TIC in FIG. 2 (b) is the result obtained by setting the flow rate of the pump 5 to 1.0 mLZmin and adding the weak base solution 3. The analysis conditions in Fig. 2 are as follows: Solution 1 contains 0.1 mol / L triethylamine acetate; 10% aqueous acetonitril solution (V / V :); Solution 2 contains 0.1 mol / L triethylamine acetate. 20% aqueous solution of acetonitrile (V / V), separation force in column 7 4.6 x 150 mm (5 μm packing material) 0 DS (otatadecylsilane) column, weak base solution 3 0.1 molZL imidazole in acetonitrile solution was used. Pump 4 has a flow rate of 0.5 mL / min, and the composition of solutions 1 and 2 starts from solution 1 solution 2 = 100 0Z 0 and takes 20 minutes to reach solution 1 Ζ solution 2 = 50Ζ50 A variable gradient elution was performed. The oligonucleic acid sample is a mixed aqueous solution of three samples with the number of bases of 20, 30, and 40. The concentrations of the 20 base sample are 24 mol / L and the 30 base sample are 40 mol / L. The L, 40 base sample was 29 μmol ZL. 30 μL of the oligonucleic acid sample was injected from the sample injector 6. By the way, the mass number (m) of the 20 base sample is 6096, that of the 30 base sample is 9200, and that of the 40 base sample is 123.60.
第 2図 ( a ) の T I Cでは、 2 0塩基試料のピークのみを検出するこ とができた。 3 0塩基試料, 4 0塩基試料はノイズ成分に埋もれてしま
い、 T I c上では特定することが出来ない。 With the TIC in Fig. 2 (a), only the peak of the 20-base sample could be detected. The 30-base sample and the 40-base sample are buried in noise components. No, it cannot be specified on TI c.
第 2図 (b ) の T I Cでは、 第 2図 ( a ) の場合に比べて相対強度が 約 1 0倍の感度向上を確認することができ、 オリ ゴ核酸試料に含まれる すべてのオリ ゴ核酸のピークが検出することができた。 In the TIC shown in Fig. 2 (b), it was confirmed that the relative intensity was about 10 times higher than that in Fig. 2 (a), and all oligo nucleic acids contained in the oligo nucleic acid sample were confirmed. Could be detected.
また、 第 2図 ( a ) および ( b ) において検出された各塩基試料ピー クの保持時間におけるマススぺク トルを第 3図に示す。 FIG. 3 shows the mass spectrum at the retention time of each base sample peak detected in FIGS. 2 (a) and (b).
弱塩基溶液 3を加えていない場合の第 3図 ( a ) のマススペク トルで は、 T I Cでピークを確認できた 2 0塩基試料は質量対電荷比 (niZ z ) 1 5 2 3に 4価の多価イオンを与えた。 また、 3 0塩基試料も mZ z 1 8 3 9の 5価の多価イオンを与えている。 一方、 弱塩基溶液 3を加え た場合の第 3図 (b ) のマススペク トルでは 2 0塩基試料、 3 0塩基試 料ともに検出感度が向上し、 S/N比が弱塩基溶液 3を加えていない場 合に比べ約 1 0倍向上した。 また弱塩基溶液 3を加えることにより、 生 成するイオンの価数が増加し、 2 0塩基試料では 5価のイオンである m ノ z 1 2 1 8のイオン、 3 0塩基試料では 6価の m/ z 1 5 3 2 と 7価 の mZ z 1 3 1 3のイオンが検出され、 より低質暈数側で検出すること ができた。 さらに、 弱塩基溶液 3を加えていない場合は全く検出できな かった 4 0塩基試料のマススぺク トルも第 4図に示したように 7価の m Z z l 7 6 4から 1 0価の m/ z 1 2 3 5のイオンまでを感度良く検出 する事ができた。 In the mass spectrum of Fig. 3 (a) without adding the weak base solution 3, the peak of 20 bases that could be confirmed by TIC was found to have a mass-to-charge ratio (niZz) of 15 Multiply charged ions were provided. The 30-base sample also gives a pentavalent polyvalent ion of mZ z 1839. On the other hand, in the mass spectrum of Fig. 3 (b) when the weak base solution 3 was added, the detection sensitivity was improved for both the 20 base sample and the 30 base sample, and the S / N ratio of the weak base solution 3 was increased. It is about 10 times higher than without. The addition of the weak base solution 3 also increases the valency of the generated ions, and the 20-base sample is a pentavalent ion, m-noz1218, and the 30-base sample is a hexavalent ion. The ions of m / z 1532 and 7-valent mZz1313 were detected, and could be detected on the lower quality number side. In addition, the mass spectrum of the 40-base sample, which was not detected at all when the weak base solution 3 was not added, was also reduced from the 7-valent mZzl 764 to the 10-valent as shown in Fig. 4. It was possible to detect ions up to m / z 1 235 with high sensitivity.
(実施例 2) (Example 2)
第 5図に、 第 2の実施例を示す。 本実施例では、 第 1の実施例の構成 に、 カラム 7からの溶出液をスプリ ッ トするスプリ ッタ 1 1を介してフ ラタシヨ ンコレクタ 1 2を接続した、 オリ ゴ核酸の分取システムの例で ある。 フラクションコレクタ 1 2もまた、 ポンプ , サンプルインジェ
クタ 6, ポンプ 5, M S' 9 と共にコントローラ 1 0で制御することが可 能である。 FIG. 5 shows a second embodiment. In this embodiment, an oligonucleic acid fractionation system in which a fraction collector 12 is connected via a splitter 11 for splitting an eluate from a column 7 to the configuration of the first embodiment is described. This is an example. Fraction collector 1 2 also pump, sample injection It can be controlled by the controller 10 together with the rectifier 6, pump 5, and MS'9.
スプリ ッタ 1 1は、 第 6図に示した構造を持ち、 内部に長さの異なる 二つの抵抗コイルを備えている。 このコイルの抵抗値の違いによって、 カラム 7からの溶出液の 1 / 1 0から 1 / 1 0 0程度の一定量を M S 9 側へ流出させ、 ポンプ 5から送られた弱塩基溶液 3 と合流させる。 こう して M S 9への流路のみに弱塩基溶液 3を合流させることが可能である c 合流した溶液はミキサ 8によって攪拌され、 MS 9に導入される。 The splitter 11 has the structure shown in FIG. 6, and has two resistance coils having different lengths inside. Due to the difference in the resistance of this coil, a certain amount of the eluate from column 7 to 1/10 to 1/1000 flows out to the MS 9 side and merges with the weak base solution 3 sent from the pump 5. Let it. In this way, the weak base solution 3 can be combined only with the flow path to the MS 9 c. The combined solution is stirred by the mixer 8 and introduced into the MS 9.
コントローラ 1 0には、 あらかじめ分取したいォリ ゴ核酸の質量数や 多価イオンの質量数を入力しておく。 MS 9 S、 これらの質量数を持つ イオンに対応したクロマ トグラムピークを検出したらコントローラ 1 0 はフラクショ ンコレクタ 1 2に信号線を通して信号を送る。 この信号を 受けて、 フラクショ ンコレクタ 1 2は目的のオリ ゴ核酸試料を試験管等 の容器に分取する。 In the controller 10, the mass number of the oligonucleic acid and the mass number of the multiply charged ions to be collected are input in advance. When MS 9 S detects a chromatogram peak corresponding to ions having these mass numbers, the controller 10 sends a signal to the fraction collector 12 through a signal line. Upon receiving this signal, the fraction collector 12 separates the desired oligo nucleic acid sample into a container such as a test tube.
また、 スプリ ッタ 1 1 とフラクショ ンコレクタ 1 2 との間に UV検出 器 (図示せず) を接続し、 UVクロマ トグラムのピーク面積値からオリ ゴ核酸試料の定量情報を得ることも可能である。 また、 UV検出器が検 出したピーク信号に基づき、 フラクションコレクタ 1 2が試料を分取す ることができる。この場合、分取と同時に M Sが分析を行っているので、 分取した試料をその質量数情報によって同定することができる。 It is also possible to connect a UV detector (not shown) between the splitter 11 and the fraction collector 12 to obtain quantitative information on oligo nucleic acid samples from the peak area of the UV chromatogram. . Further, the fraction collector 12 can sample the sample based on the peak signal detected by the UV detector. In this case, since the MS is performing analysis simultaneously with the fractionation, the fractionated sample can be identified by its mass number information.
また、 第 2の実施例において、 オリ ゴ核酸試料の量が少なく M S 9で の検出が困難な場合、 または第 6図のスプリ ツタ 1 1で試料溶液を分け ることすら困難な場合は、 第 7図に示すよ うに、 一定時間間隔で実線流 路と点線流路を交互に切替える六方パルプ 1 3をカラム 7 と MS 9およ ぴフラクシヨ ンコレクタ 1 2間に接続する。 カラム 7からの溶出液は、
六方パルプ 1 3の切替えによって M S 9 とフラクショ ンコレクタ 1 2 と に交互に流出する。 第 2図に示すように、 1成分のオリ ゴ核酸試料のピ 一ク幅は約 3 0秒程度あるため、 六方バルブ 1 3の流路の切替えは、 例 えば 1秒間隔で行うようにする。 In the second embodiment, when the amount of the oligonucleic acid sample is small and it is difficult to detect the sample with MS9, or when it is difficult even to separate the sample solution with the splitter 11 in FIG. As shown in Fig. 7, a hexagonal pulp 13 that alternates between a solid flow path and a dotted flow path at regular time intervals is connected between the column 7 and the MS 9 and the fraction collector 12. The eluate from column 7 is When the hexagonal pulp 13 is switched, it flows out alternately to the MS 9 and the fraction collector 12. As shown in Fig. 2, since the peak width of the one-component oligonucleic acid sample is about 30 seconds, the flow path of the hexagonal valve 13 should be switched, for example, every 1 second. .
カラム 7からの溶出液が M S 9へ流出する場合は、 一度、 六方パルプ 1 3内のサンプルループ 1 3 0に保持され、 その後ポンプ 5の送る弱塩 基溶液 3によって M S .9へ送られる。 When the eluate from the column 7 flows out to the MS 9, it is once held in the sample loop 130 in the hexagonal pulp 13, and then sent to the MS 9 by the weak base solution 3 sent from the pump 5.
第 7図の構成においても、 第 5図の例と同様に、 M S 9が目的とする ォリ ゴ核酸試料の質量数を持つイオンに対応したク口マトグラムピーク を検出すると、 コントローラ 1 0はフラクションコレクタ 1 2に信号を 送り、 フラクションコレクタ 1 2は目的のオリ ゴ核酸試料を分取する。 In the configuration of FIG. 7 as well, as in the example of FIG. 5, when the MS 9 detects a mouth massogram peak corresponding to an ion having the mass number of the target oligonucleic acid sample, the controller 10 returns to the controller 10. A signal is sent to the fraction collector 12, which collects the target oligonucleic acid sample.
(実施例 3 ) (Example 3)
第 8図に、 第 3の実施例を示す。 本実施例では、 第 1の実施例の構成 に対して、 カラムを追加し、 二種類のオリ ゴ核酸試料を同時に分析でき るように拡張した例である。 追加流路であるカラム 1 5, ミキサ 1 6以 外に、 カラム 7, 1 5からの二つの流路に弱塩基溶液 3を等分するため のスプリ ッタ 1 7, カラム 7, 1 5の出口からの二つの流路を一つの M Sへ導入するための切替えを行う十方バルブ 1 8 , 十方パルプ 1 8に 到達した溶出液を M S 9へ送るための移送液 1 9、 およぴ送液ポンプ 2 0を追加している。十方パルプ 1 8 とポンプ 2 0も、ポンプ 4 と 1 4, サンプルインジヱクタ 6, ポンプ 5, M S 9 と同一のコントローラ 1 0 で制御可能である。 また、 サンプルインジェクタ 6はいずれの流路に対 しても試料を注入することが可能である。 FIG. 8 shows a third embodiment. This embodiment is an example in which a column is added to the configuration of the first embodiment so that two types of oligonucleic acid samples can be simultaneously analyzed. In addition to columns 15 and mixer 16 as additional flow paths, a splitter 17 for equally dividing weak base solution 3 into two flow paths from columns 7 and 15 and a flow path for columns 7 and 15 A 10-way valve 18 for switching the two flow paths from the outlet to one MS, a transfer solution 19 for sending the eluate reaching the 10-way pulp 18 to the MS 9, and Liquid sending pump 20 is added. The 10-way pulp 18 and the pump 20 can also be controlled by the same controller 10 as the pumps 4 and 14, the sample injector 6, the pump 5, and the MS9. The sample injector 6 can inject a sample into any of the flow paths.
サンプルィンジヱクタ 6より二つの流路に注入された二種類のオリ ゴ 核酸試料は、 それぞれのカラム 7, 1 5により単一成分に分離される。
ポンプ 5によつて送られた弱塩基溶液 3は、 スプリ ツタ 1 7によつて二 つの流路に等量に分けられる。 分けられたそれぞれの弱塩基溶液 3は、 カラム 7, 1 5からの溶出液とそれぞれ合流し、 ミキサ 8, 1 6によつ て攪拌され十方パルプ 1 8に送られる。 十方パルプ 1 8は一定時間間隔 (例えば 1秒) で実線流路と点線流路を切替え、 ポンプ 2 0の送る移送 液 1 9によって MS 9に対して二つのカラムからの溶出液が交互に導入 される。 MS 9はバルブ 1 8の切替え時間間隔に基づき、 検出した信号 が二つの流路のどちらに起因するものかを判断し、,'二種類の信号を別個 に処理する。 これにより、 一台の MS 9で二つのカラム 7 , 1 5から溶 出したオリ ゴ核酸試料の質量情報を得ることができる。 The two types of oligonucleic acid samples injected into the two channels from the sample injector 6 are separated into single components by the respective columns 7 and 15. The weak base solution 3 sent by the pump 5 is divided into two equal volumes by the splitter 17. Each of the divided weak base solutions 3 is combined with the eluate from the columns 7 and 15, respectively, and stirred by the mixers 8 and 16 to be sent to the 10-way pulp 18. The 10-way pulp 18 switches between the solid flow path and the dotted flow path at regular time intervals (for example, 1 second). be introduced. The MS 9 determines, based on the switching time interval of the valve 18, whether the detected signal is due to one of the two flow paths, and processes the two types of signals separately. As a result, mass information of the oligonucleic acid sample dissolved from the two columns 7 and 15 can be obtained by one MS 9.
こ こで移送液 1 9は、 二つの流路からの溶出液を送る役割と同時に、 M Sのイオン源内での二つの溶出液の混合を防ぐ役割を持つ。 したがつ て移送液 1 9は、 MS 9での分析に影響を与えないものが良く、 既に溶 離液に加えられている弱塩基溶液 3を用いることが MS 9の感度を上げ ることもでき、 最.も良い結果を与える。 弱塩基溶液 3以外ではメタノー ル等を用いることが出来る。 Here, the transfer solution 19 has a role of sending the eluate from the two flow paths and a role of preventing the mixing of the two eluate in the MS ion source. Therefore, it is preferable that the transfer solution 19 does not affect the analysis in the MS 9, and the use of the weak base solution 3 already added to the eluate may increase the sensitivity of the MS 9 Can do the best. Other than the weak base solution 3, methanol or the like can be used.
第 9図及び第 1 0図に本実施例の変形例を示す。 9 and 10 show a modification of the present embodiment.
第 9図の例は、 スプリ ッタ 1 1 と 2 1、 一つのコントローラ 1 0で制 御可能なフラクションコレクタ 1 2, 2 2を接続し、 流路の数に応じた オリ ゴ核酸試料分取システム'を実現するものである。 スプリ ッタ 1 1, 2 1は、 第 6図で示したものを使用する。 In the example of Fig. 9, the splitters 11 and 21 and the fraction collectors 12 and 22 that can be controlled by one controller 10 are connected, and the oligonucleic acid sample collection according to the number of flow paths is performed. System '. Use the splitters 11 and 21 shown in Fig. 6.
また、 第 1 0図の例は、 オリ ゴ核酸試料の量が少ない場合の概略構成 図である。 スプリ ッタ 1 1 , 2 1を用いずに、 カラム 7, 1 5からの溶 出液の全量に弱塩基溶液 3を合流させて、 十方バルブ 1 8によってカラ ム 7, 1 5から溶出した試料のうち M S 9に導入するもの以外のオリ ゴ
核酸試料を全量分取するものである。 Further, the example of FIG. 10 is a schematic configuration diagram when the amount of the oligonucleic acid sample is small. Without using splitters 11 and 21, weak base solution 3 was combined with all of the eluate from columns 7 and 15 and eluted from columns 7 and 15 by 10-way valve 18. Samples other than those to be introduced into MS 9 A nucleic acid sample is collected in its entirety.
第 9図及ぴ第 1 0図の例によれば、 容量の少ない試料の場合でも、 M Sで分析する用途以外は、 漏れなく分取することが出来る。 According to the examples shown in FIGS. 9 and 10, even if the sample has a small capacity, it can be collected without leakage except for the purpose of analyzing by MS.
上記に示した各実施例に拠れば、 L Cからの溶出液がオンライ ンで M Sへ導入される L C Z M Sを用いて、 オリ ゴ核酸の高感度分析が可能 になる。 従って、 分析の効率を向上させることが可能となる。 According to each of the above-described examples, highly sensitive analysis of oligonucleic acid can be performed using LCZMS in which eluate from LC is introduced online to MS. Therefore, it is possible to improve the efficiency of the analysis.
さらに、 弱塩基溶液の作用により、 多価イオンが生成しやすくなるた め、 T〇 F— M Sより も測定可能質量数の上限が低いイオントラップ型 や四重極型の質量分析装置での測定が可能になる。 また、 T O F— M S を用いた分析においても分析の効率を大きく上げる事ができる。 Furthermore, the action of a weak base solution facilitates the generation of multiply charged ions, so measurement with an ion trap type or quadrupole type mass spectrometer, which has a lower upper limit of the measurable mass number than T〇F-MS Becomes possible. In addition, the efficiency of the analysis using TOF-MS can be greatly increased.
また、 マススぺク トルが感度良く検出できるようになるため、 目的と するオリ ゴ核酸の質量数や、 その多価イオンの質量数を信号と して、 ォ リ ゴ核酸の分取を行う事が可能になる。 この時、 マススペク トルを取り ながら分取を行うため、 分取したオリ ゴ核酸試料のマススぺク トルを試 料の品質データと して分離と同時に記録することが可能である。 In addition, since the mass spectrum can be detected with high sensitivity, the oligonucleic acid is fractionated using the mass number of the target oligonucleic acid and the mass number of the polyvalent ion as a signal. Becomes possible. At this time, since the fractionation is performed while taking the mass spectrum, it is possible to record the mass spectrum of the fractionated oligonucleic acid sample simultaneously with the separation as quality data of the sample.
さらに、 同時に複数のカラムによる分離を行いながら、 それより少な い M Sによる複数のオリ ゴ核酸の高感度分析および分取が可能になる。 また、 M Sによる複数のオリ ゴ核酸の分析時に、 切替えパルプから M S への送液を独立したポンプで行うため、 試料同士のィオン源内での混合 を防ぐ事ができる。
In addition, it enables high-sensitivity analysis and fractionation of multiple oligonucleic acids with less MS while simultaneously performing separation on multiple columns. In addition, when a plurality of oligonucleic acids are analyzed by the MS, the liquid is transferred from the switched pulp to the MS using an independent pump, so that mixing of the samples in the ion source can be prevented.
Claims
1 . イオンペア剤や塩を含む溶離液を送液するポンプ、 測定対象試料を 流路内に注入するサンプルィンジェクタ、 導入された溶液を成分ごとに 分離する力ラム、 当該カラムから溶出してきた溶液を大気圧イオン源に よってイオン化し検出する質量分析装置を有する液体ク口マ トグラフ質 量分析装置であって、 1. A pump that sends an eluent containing ion-pairing agents and salts, a sample injector that injects the sample to be measured into the flow channel, a power ram that separates the introduced solution for each component, and eluted from the column A liquid chromatograph mass spectrometer having a mass spectrometer for ionizing and detecting a solution with an atmospheric pressure ion source,
前記カラムと前記質量分析装置の間に、 弱塩基の溶液を合流させる合 流手段を備えたことを特徴とする液体ク口マトグラフ質量分析装置。 Liquid mass chromatograph mass spectrometer characterized by comprising a merging means for merging a weak base solution between the column and the mass spectrometer.
2 . 請求項 1において、 2. In Claim 1,
前記合流後の流路に、 溶液を攪拌, 混合するミキシング手段を備えた ことを特徴とする液体ク口マトグラフ質量分析装置。 A liquid mouth mass spectrometer characterized by comprising a mixing means for stirring and mixing the solution in the flow path after the merging.
3 . 請求項 2において、 3. In Claim 2,
前記力ラムと前記ミキシング手段の間に、 力ラムから溶出した溶液の 流れを所定の割合で分岐させる分岐手段を備え、 当該溶液の一部を前記 質量分析装置に、 その他の溶液をフラクシヨ ンコレクタに導入するよう にしたことを特徴とする液体ク口マトグラフ質量分析装置。 A branching means for branching a flow of the solution eluted from the force ram at a predetermined ratio between the force ram and the mixing means, a part of the solution to the mass spectrometer, and the other solution to a fraction collector Liquid mass chromatography mass spectrometer characterized by being introduced.
4 . 請求項 3において、 4. In Claim 3,
前記合流手段は前記分岐手段に接続され、 前記質量分析装置に導入す る溶液に対して前記弱塩基の溶液を混合させることを特徴とする液体ク 口マトグラフ質量分析装置。 A liquid chromatograph mass spectrometer, wherein the merging unit is connected to the branching unit, and mixes the solution of the weak base with a solution introduced into the mass spectrometer.
5 . 請求項 3において、 5. In Claim 3,
前記分岐手段は、 六方バルブであり、 内部に」時的に溶液を保持する サンプルループを備え、 所定の周期で流路を切替えることを特徴とする 液体クロマ トグラフ質量分析装置。 The liquid chromatograph mass spectrometer is characterized in that the branching means is a six-way valve, includes a sample loop therein for temporarily holding the solution, and switches a flow path at a predetermined cycle.
6 . 請求項 3において、
前記フラクショ ンコレクタは、 前記質量分析装置で検出された信号に 応じて動作することを特徴とする液体ク口マトグラフ質量分析装置。 6. In Claim 3, The said fraction collector operates according to the signal detected by the said mass spectrometry apparatus, The liquid chromatograph mass spectrometer characterized by the above-mentioned.
7 . 請求項 3において、 7. In Claim 3,
前記分岐手段とフラクションコレクタとの間に、 U V検出器を備えた ことを特徴とする液体ク口マトグラフ質量分析装置。 A liquid chromatograph mass spectrometer comprising a UV detector between the branching means and the fraction collector.
8 . 請求項 1において、 8. In claim 1,
質量分析装置は、 エレク トロスプレイイオン化法, ソニックスプレイ イオン化法及びイオンスプレイでイオン化することを特徴とする液体ク 口マトグラフ質量分析装置。 The mass spectrometer is an electrospray ionization method, a sonic spray ionization method, and ionization by ion spray.
9 . 請求項 1 において、 9. In Claim 1,
質量分析装置は、 飛行時間型, イオントラップ型、 または四重極型の 質量分析装置であることを特徴とする液体クロマトグラフ質量分析装置 ( 1 0 . イオンペア剤や塩を含む溶離液を送液する溶離液用ポンプ, 測定 対象試料を流路内に注入するサンプルィンジェクタ、 導入された溶液を 成分ごとに分離する複数のカラム、 当該力ラムから溶出してきた溶液を 大気圧イオン源によってイオン化し検出する質量分析装置を有する液体 クロマ トダラフ質量分析装置であって、 The mass spectrometer is a time-of-flight, ion trap, or quadrupole mass spectrometer. Liquid chromatography mass spectrometer (10. Sends eluent containing ion pairing agent or salt) Eluent pump, sample injector for injecting the sample to be measured into the flow channel, multiple columns to separate the introduced solution for each component, and solution eluted from the power ram to be ionized by the atmospheric pressure ion source A liquid chromatograph mass spectrometer having a mass spectrometer for detecting and detecting
前記各力ラムの下流の流路のそれぞれに設けられた溶液を攪拌, 混合 するミキシング手段と、 Mixing means for stirring and mixing the solution provided in each of the flow paths downstream of the respective force rams;
前記カラムと前記ミキシング手段の間に弱塩基の溶液を合流させる合 流手段と、 Merging means for merging a solution of a weak base between the column and the mixing means;
前記各ミキシング手段からの溶液が導かれ、 且つ前記質量分析装置と 接続された切替えバルブと、 A switching valve through which the solution from each of the mixing means is guided, and which is connected to the mass spectrometer;
前記切替えパルプに対して移送液を送液する移送液用ポンプとを備え. 前記切替えバルブを切替えながら、 前記移送液によって前記切替えバ
ルブに導かれた前記各力ラムからの溶出液を交互に前記質量分析装置に 導入することを特徴とする液体ク口マ トグラフ質量分析装置。 And a transfer liquid pump for sending a transfer liquid to the switching pulp. The switching valve is switched by the transfer liquid while switching the switching valve. A liquid chromatograph mass spectrometer, wherein an eluate from each of the force rams led to a lube is alternately introduced into the mass spectrometer.
1 1 . 請求項 1 0において、 1 1. In claim 10,
前記移送液は、 弱塩基の溶液を用いることを特徴とする液体ク口マト グラフ質量分析装置,。 A liquid chromatograph mass spectrometer, wherein the transfer liquid uses a solution of a weak base.
1 2 . 請求項 1 0において、 1 2. In claim 10,
前記カラムの数と同数のフラクショ ンコレクタを備え、 The same number of fraction collectors as the number of columns,
前記力ラムと前記ミキシング手段の間のそれぞれの流路に、 カラムか ら溶出した溶液の流れを所定の割合で分岐させる分岐手段を備え、 当該複数の分岐手段は、 前記切替えバルブと各フラクショ ンコ レクタ に接続されることを特徴とする液体ク口マトグラフ質量分析装置。 In each flow path between the force ram and the mixing means, there is provided a branch means for branching a flow of the solution eluted from the column at a predetermined ratio, and the plurality of branch means are provided with the switching valve and the respective fraction switches. Liquid mass chromatography mass spectrometer characterized by being connected to a collector.
1 3 . 請求項 1 2において、 1 3. In claim 12,
前記合流手段は前記各分岐手段に接続され、 前記切替えパルプに導入 する溶液に対して前記弱塩基の溶液を混合させることを特徴とする液体 ク ロマ トグラフ質量分析装置。 A liquid chromatograph mass spectrometer, wherein the merging unit is connected to each of the branching units, and mixes the solution of the weak base with the solution introduced into the switching pulp.
1 4 . 請求項 1 0において、 14. In claim 10,
前記切替えパルプに接続され、 且つ前記力ラムの数と同数のフラク シ ヨンコレクタを備えたことを特徴とする液体ク口マトグラフ質量分析装
A liquid mouth mass spectrometer which is connected to the switching pulp and has the same number of fraction collectors as the number of the power rams.
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JP2006292542A (en) * | 2005-04-11 | 2006-10-26 | Hitachi High-Technologies Corp | Liquid chromatograph mass spectrometer |
WO2025013534A1 (en) * | 2023-07-07 | 2025-01-16 | 株式会社島津製作所 | Analysis method, evaluation method, and program |
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