JP5054439B2 - Two-dimensional liquid chromatograph with ion exchange and normal phase column - Google Patents

Two-dimensional liquid chromatograph with ion exchange and normal phase column Download PDF

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JP5054439B2
JP5054439B2 JP2007158899A JP2007158899A JP5054439B2 JP 5054439 B2 JP5054439 B2 JP 5054439B2 JP 2007158899 A JP2007158899 A JP 2007158899A JP 2007158899 A JP2007158899 A JP 2007158899A JP 5054439 B2 JP5054439 B2 JP 5054439B2
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dimensional liquid
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ion exchange
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JP2008309699A (en
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公良 甲田
喜三郎 出口
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Hitachi High Tech Corp
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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
    • B01D15/30—Partition chromatography
    • B01D15/305—Hydrophilic interaction chromatography [HILIC]
    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B01D15/1878—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series for multi-dimensional chromatography
    • 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
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange

Description

本発明は、液体クロマトグラフィ装置に係わり、特に、順相及びイオン交換分離カラムを有する順相型２次元液体クロマトグラフィ装置に関する。   The present invention relates to a liquid chromatography apparatus, and more particularly to a normal phase type two-dimensional liquid chromatography apparatus having normal phase and ion exchange separation columns.

生体試料のような複雑なサンプルの分離には、多次元液体クロマトグラフィ（ｎＤ−ＨＰＬＣ）が有効である。プロテオミックスの分野では、イオン交換（ＩＥＸ）―逆相（ＲＰ）カラムを連結した２次元液体クロマトグラフィ（２Ｄ−ＨＰＬＣ）で分離し、質量分析計（ＭＳ）でオンライン分析する方法および装置がよく使われている（非特許文献１）。   Multidimensional liquid chromatography (nD-HPLC) is effective for separating complex samples such as biological samples. In the field of proteomics, a method and an apparatus are often used for separation by two-dimensional liquid chromatography (2D-HPLC) coupled with an ion exchange (IEX) -reverse phase (RP) column and on-line analysis with a mass spectrometer (MS). (Non-Patent Document 1).

一方、親水性の大きい成分（例えば、糖鎖や糖ペプチド）の分離分析では、上記の逆相型２次元液体クロマトグラフィの適用が困難である。従って、イオン交換（ＩＥＸ）―逆相（ＲＰ）―順相（ＮＰ／ＨＩＬＬＩＣ）カラムを組み合わせた３次元液体クロマトグラフィ（２Ｄ−ＨＰＬＣ）が、最も分離能力が高く、よく用いられてきた（図１参照）（非特許文献２）。しかし、逆相モードと順相モードではグラヂエント溶出溶媒の組成が逆転するために（“溶離液の干渉”とも言われる）、分取、脱溶媒および濃縮、再注入を繰り返すオフライン処理が必要となり、時間と労力がかかるだけでなく、サンプルの損失や検出感度の低下を招く恐れがある。   On the other hand, it is difficult to apply the above-described reversed-phase two-dimensional liquid chromatography in the separation and analysis of highly hydrophilic components (for example, sugar chains and glycopeptides). Therefore, three-dimensional liquid chromatography (2D-HPLC) combining an ion exchange (IEX) -reverse phase (RP) -normal phase (NP / HILLIC) column has the highest resolution and has been frequently used (FIG. 1). (See Non-Patent Document 2). However, since the composition of the gradient elution solvent is reversed between the reverse phase mode and the normal phase mode (also referred to as “eluent interference”), an offline process that repeats fractionation, desolvation and concentration, and reinjection is required. Not only is it time consuming and labor intensive, but there is a risk of losing samples and reducing detection sensitivity.

Ａ．Ｊ．Ｌｉｎｋ，ｅｔ．ａｌ．，Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．１７（１９９９）６７６．A. J. et al. Link, et. al. Nat. Biotechnol. 17 (1999) 676. Ｔ．Ｔａｋａｈａｓｈｉ，ｅｔ．ａｌ．，Ａｎａｌ．Ｂｉｏｃｈｅｍ．２２６（１９９５）１３９．T.A. Takahashi, et. al. , Anal. Biochem. 226 (1995) 139. Ｍ．Ａ．Ｓｔｒｅｇｅ，ｅｔ．ａｌ．，Ａｎａｌ．Ｃｈｅｍ．７２（２０００）４６２９．M.M. A. Strage, et. al. , Anal. Chem. 72 (2000) 4629.

本発明の目的は、イオン交換（ＩＥＸ）―順相（ＮＰ／ＨＩＬＩＣ）カラムを直列に組み合わせた２Ｄ−ＨＰＬＣを順相（ヒリック）モードで用いることにより、上記の従来型技術である逆相型２Ｄ−ＨＰＬＣや３Ｄ−ＨＰＬＣの問題点を解決することである。   The object of the present invention is to use the 2D-HPLC in which the ion exchange (IEX) -normal phase (NP / HILIC) column is combined in series in the normal phase (Hylic) mode, so that the above-mentioned conventional technique, the reverse phase type is used. It is to solve the problems of 2D-HPLC and 3D-HPLC.

一般に、ＩＥＸカラムは水系で用いられるので、ＲＰカラムとの相性がよい。しかし、ＩＥＸカラムがアセトニトリル溶媒（５０％以上）、つまり、ＨＩＬＬＩＣモードでも機能することはあまり知られていなかった（非特許文献３）。本発明は、この点に注目してＩＥＸ−ＨＩＬＬＩＣカラムを連結したオンライン２Ｄ（ＩＥＸ−ＨＩＬＬＩＣ）ＨＰＬＣシステムで親水性の高い成分（例えば、糖鎖や糖ペプチド）の分離分析の実現を図るものである。   In general, since an IEX column is used in an aqueous system, it is compatible with an RP column. However, it has not been well known that the IEX column functions even in acetonitrile solvent (50% or more), that is, in the HILLIC mode (Non-patent Document 3). The present invention focuses on this point and realizes separation and analysis of highly hydrophilic components (for example, sugar chains and glycopeptides) using an on-line 2D (IEX-HILLIC) HPLC system connected with an IEX-HILLIC column. is there.

本発明は、特に、生体内に存在する親水性物質を分析する、液体クロマトグラフ装置において、イオン交換と順相（／ヒリック）分離モードを組み合わせて分離分析を行うものに適する２次元液体クロマトグラフィ装置を提供するものである。   The present invention particularly relates to a two-dimensional liquid chromatographic apparatus suitable for a liquid chromatographic apparatus for analyzing a hydrophilic substance present in a living body and performing separation analysis by combining ion exchange and normal phase (/ hilic) separation mode. Is to provide.

より具体的には、本発明は、複数の溶液を混合し送液する手段（例えばグラジエントポンプ）と、試料注入手段と、前段分離カラム（例えばイオン交換を行う）および後段分離カラム（順相／ヒリック（親水性相互作用））と、分離された成分を検出する一つまたは複数の手段から構成される液体クロマトグラフ装置において、イオン交換と順相（／ヒリック）分離モードを組み合わせて分離分析を行うことを特徴とする２次元液体クロマトグラフィ装置を提供するものである。   More specifically, the present invention relates to a means for mixing and feeding a plurality of solutions (for example, a gradient pump), a sample injection means, a front separation column (for example, performing ion exchange), and a rear separation column (normal phase / HILIC (hydrophilic interaction)) and a liquid chromatograph consisting of one or more means for detecting separated components. Separation analysis is performed by combining ion exchange and normal phase (/ HIRIC) separation mode. The present invention provides a two-dimensional liquid chromatography apparatus.

本発明によれば、サンプル中の非イオン性、イオン性の親水性成分（例えば、糖鎖や糖ペプチド）の一斉分離分析をオンラインで実現できる。   According to the present invention, simultaneous separation analysis of nonionic and ionic hydrophilic components (for example, sugar chains and glycopeptides) in a sample can be realized online.

本発明の好ましい実施形態としては、前記２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオン、または陰イオン交換カラムであることを特徴とする２次元液体クロマトグラフィ装置がある。また、前記２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオンと陰イオン交換充填剤を１本のカラムに充填したものであることを特徴とする２次元液体クロマトグラフィ装置がある。更に、前記２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオンと陰イオン交換充填剤を１本のカラムに充填し、陽（陰）イオンと陰（陽）イオン交換カラムを直列に接続したものであることを特徴とする２次元液体クロマトグラフィ装置もある。   As a preferred embodiment of the present invention, in the two-dimensional liquid chromatography apparatus, there is a two-dimensional liquid chromatography apparatus characterized in that the pre-stage separation column is a cation or anion exchange column. In the two-dimensional liquid chromatography apparatus, there is a two-dimensional liquid chromatography apparatus characterized in that the pre-stage separation column is one in which a cation and an anion exchange packing material are packed in one column. Further, in the two-dimensional liquid chromatography apparatus, the first separation column is filled with a cation and an anion exchange packing material in one column, and the cation (anion) and the anion (cation) ion exchange column are connected in series. There is also a two-dimensional liquid chromatography apparatus characterized by the above.

そして、前記２次元液体クロマトグラフィ装置において、検出手段として質量分析計を用いることを特徴とする２次元液体クロマトグラフィ装置がある。   In the two-dimensional liquid chromatography apparatus, there is a two-dimensional liquid chromatography apparatus using a mass spectrometer as a detecting means.

以下に、本発明の実施形態を添付図面を参照しながら説明する。   Embodiments of the present invention will be described below with reference to the accompanying drawings.

図１はプロテオミックスで一般的に用いられている２次元液体クロマトグラフィ装置の構成を示し、図２はイオン交換（ＤＥＡＥ），逆相、順相（ＮＰ／ＨＩＬＩＣ）カラムを組み合わせた、従来のオフライン３次元液体クロマトグラフィ装置の構成を示す。   Fig. 1 shows the configuration of a two-dimensional liquid chromatography apparatus commonly used in proteomics, and Fig. 2 shows a conventional offline combination of ion exchange (DEAE), reverse phase, and normal phase (NP / HILIC) columns. The structure of a three-dimensional liquid chromatography apparatus is shown.

図３は、本発明の第１の実施形態であり、最も単純な順相型２次元液体クロマトグラフィ装置の構成である。各構成ユニットの機能と動作原理を以下に記述する。   FIG. 3 shows the first embodiment of the present invention, which is the simplest normal phase type two-dimensional liquid chromatography apparatus. The functions and operating principles of each component unit are described below.

ステップ１：ポンプは、水溶液Ａおよび有機溶媒溶液Ｂを混合（Ｂの組成比が高い）し、一定流量で送液を行う。試料注入装置（ＡＳ）は、一定量の試料を高圧流路に注入する。   Step 1: The pump mixes the aqueous solution A and the organic solvent solution B (the composition ratio of B is high), and sends the solution at a constant flow rate. The sample injection device (AS) injects a certain amount of sample into the high-pressure channel.

ステップ２：注入された試料中のイオン性の成分は前段のイオン交換（ＩＥＸ）カラムで保持され、非イオン性の成分は後段の順相（ＨＩＬＩＣ）カラムに保持される。   Step 2: The ionic component in the injected sample is retained in the former ion exchange (IEX) column, and the non-ionic component is retained in the latter normal phase (HILIC) column.

ステップ３：順相（ＨＩＬＬＩＣ）カラムに保持された成分を、次第に有機溶媒液Ｂの組成比を下げて送液することにより、分離、溶出させる（ＨＩＬＩＣモードでのグラジエント溶出）。 Step 3: The components held in the normal phase (HILIC) column are separated and eluted by gradually reducing the composition ratio of the organic solvent liquid B (gradient elution in the HILIC mode).

ステップ４：高塩濃度水溶液Ｃと有機溶媒溶液Ｂを混合し、一定時間送液することにより、前段のイオン交換（ＩＥＸ）カラムに保持されていた成分を溶出させる。溶出した成分は後段の順相（ＨＩＬＬＩＣ）カラムに保持される。   Step 4: The high salt concentration aqueous solution C and the organic solvent solution B are mixed and sent for a certain period of time to elute the components retained in the previous ion exchange (IEX) column. The eluted components are retained in the latter normal phase (HILLIC) column.

ステップ５：ステップ３に戻る。   Step 5: Return to Step 3.

ステップ６：ステップ４−５を繰り返す。   Step 6: Repeat Step 4-5.

図４は、ヒト血清中の２−アミノピリジン（ＰＡ）誘導体化糖鎖を上記の方法で分離分析したクロマトグラムである。検出手段としては、蛍光検出器（励起波長：３００ｎｍ、蛍光波長：４２０ｎｍ）を使用した。また、イオン交換カラム（内径２ｍｍ、長さ７５ｍｍ）としてはＤＥＡＥ（デエチルアミノエタン）型を、また、ＨＩＬＩＣカラム（内径２ｍｍ、長さ１５０ｍｍ）としてはツヴィッターイオン（ＺＩＣ）型を用いている。ポンプ流量（０．２ｍｌ／ｍｉｎ）は一定である。最初の（０−６５）分までの１回目のＨＩＬＬＩＣグラジエント溶出（水／ＡＣＮ＝２０／８０３５／６５）では、中性糖鎖とＩＥＸカラムでの保持が弱いモノシアリル糖鎖が溶出し分離される（ステップ３）。（６５−７０）分間、５００ｍＭ酢酸アンモニューム（２５％）を送液することにより（ステップ４）、保持が強いジシアリル糖鎖はＤＥＡＥカラムからＺＩＣ−ＨＩＬＬＩＣカラムに移動し、（７０−１１０）分間の２回目のＨＩＬＬＩＣグラジエント溶出（水／ＡＣＮ＝２５／７５ ３５／６５）で分離分析される（ステップ５）。同様に、保持が強いトリ、テトラシアリル糖鎖も、順次分離分析が可能である（ステップ６）。このように、全体の分離分析時間は１８０分と長いが、本方法では一回の試料注入で中性からテトラシアリル糖鎖までを一斉分離することが可能となる。   FIG. 4 is a chromatogram obtained by separating and analyzing 2-aminopyridine (PA) derivatized sugar chains in human serum by the above method. As a detection means, a fluorescence detector (excitation wavelength: 300 nm, fluorescence wavelength: 420 nm) was used. Further, a DEAE (deethylaminoethane) type is used as the ion exchange column (inner diameter 2 mm, length 75 mm), and a Zwitter ion (ZIC) type is used as the HILIC column (inner diameter 2 mm, length 150 mm). . The pump flow rate (0.2 ml / min) is constant. In the first HILLIC gradient elution (water / ACN = 20/8035/65) up to the first (0-65) minutes, neutral glycans and monosialyl glycans that are weakly retained on the IEX column are eluted and separated. (Step 3). By feeding 500 mM ammonium acetate (25%) for (65-70) minutes (step 4), the strongly retained disialyl sugar chain moves from the DEAE column to the ZIC-HILLIC column, and (70-110) minutes Are separated and analyzed by the second HILLIC gradient elution (water / ACN = 25/75 35/65) (step 5). Similarly, tri- and tetra-sialyl sugar chains with strong retention can be sequentially separated and analyzed (step 6). As described above, although the total separation analysis time is as long as 180 minutes, the present method enables simultaneous separation from neutral to tetrasialyl sugar chains by a single sample injection.

図５は、本発明の第２の実施形態である。図３の第１の実施形態との違いは、１０方流路切り替えバルブと順相（ＨＩＬＬＩＣ）トラップカラムＡ，Ｂを追加し、２台のポンプでＩＥＸ分離カラムおよびＨＩＬＩＣトラップ＆ＨＩＬＬＩＣ分離カラムに最適な溶液組成の送液を行えるようにしたものである。各構成ユニットの機能と動作原理を以下に記述する。   FIG. 5 is a second embodiment of the present invention. The difference from the first embodiment of FIG. 3 is that a 10-way flow switching valve and a normal phase (HILLIC) trap column A and B are added, and two pumps are optimal for IEX separation column and HILIC trap & HILLIC separation column. In this way, it is possible to send a solution having a proper solution composition. The functions and operating principles of each component unit are described below.

ステップ１：ポンプ１は、水溶液Ａおよび有機溶媒溶液Ｂを混合（Ｂの組成比が高い）し、一定流量で送液を行う。試料注入装置（ＡＳ）は、一定量の試料を流路に注入する。   Step 1: The pump 1 mixes the aqueous solution A and the organic solvent solution B (the composition ratio of B is high), and sends the solution at a constant flow rate. The sample injection device (AS) injects a certain amount of sample into the flow path.

ステップ２：注入された試料中のイオン性の成分は前段のイオン交換（ＩＥＸ）カラムで保持され、非イオン性の成分は１０方バルブのＨＩＬＩＣトラップカラムＡに、一端保持される。   Step 2: The ionic component in the injected sample is held in the previous ion exchange (IEX) column, and the non-ionic component is held in the HILIC trap column A having a 10-way valve.

ステップ３：１０方バルブの流路を切り替えて、ＨＩＬＬＩＣトラップカラムＡに保持された成分を、ポンプ２において次第に有機溶媒溶液Ｂの組成比下げて送液することにより、分離、溶出させる（ＨＩＬＬＩＣモードでのグラジエント溶出）。この間に、ポンプ１は高塩濃度水溶液Ｃと有機溶媒溶液Ｂを混合し、一定時間送液することにより、前段のイオン交換（ＩＥＸ）カラムに保持されていた成分を溶出させ、もう一方のトラップカラムＢに保持させる。その後、水溶液Ａおよび有機溶媒溶液Ｂを混合（Ｂの組成比が高い）した溶液を、一定時間、送液を行い、トラップカラムＢ中の塩（高塩濃度水溶液Ｃ）を洗い流す。   Step 3: By switching the flow path of the 10-way valve, the components held in the HILLIC trap column A are separated and eluted by gradually reducing the composition ratio of the organic solvent solution B in the pump 2 (HILLIC mode). Gradient elution). During this time, the pump 1 mixes the high salt concentration aqueous solution C and the organic solvent solution B, and sends the solution for a certain period of time, thereby eluting the component retained in the previous ion exchange (IEX) column and the other trap. Hold in column B. Thereafter, a solution obtained by mixing the aqueous solution A and the organic solvent solution B (the composition ratio of B is high) is fed for a certain period of time to wash away the salt (high salt concentration aqueous solution C) in the trap column B.

ステップ４：ステップ３を繰り返す。   Step 4: Repeat step 3.

この第２の実施形態の利点は、イオン交換カラムからの溶出し使用する、塩濃度が高い溶液（Ｃ）を、ＨＩＬＬＩＣ分離カラムに導入しないようにできる点である。本方法は、検出器として質量分析計（ＭＳ）を用いる場合、検出感度、装置のメインテナンス上有効である。ただし、ＨＩＬＬＩＣトラップカラムＡ，Ｂで保持が弱い成分はドレインに流出してしまうといった欠点がある。   The advantage of this second embodiment is that the solution (C) having a high salt concentration eluted and used from the ion exchange column can be prevented from being introduced into the HILLIC separation column. This method is effective in terms of detection sensitivity and apparatus maintenance when a mass spectrometer (MS) is used as a detector. However, there is a drawback that components that are weakly held in the HILLIC trap columns A and B flow out to the drain.

プロテオミックスで一般的に用いられている２次元液体クロマトグラフィ装置の構成を示す。The structure of the two-dimensional liquid chromatography apparatus generally used by proteomics is shown. イオン交換（ＤＥＡＥ），逆相、順相（ＮＰ／ＨＩＬＩＣ）カラムを組み合わせた、従来のオフライン３次元液体クロマトグラフィ装置の構成を示す。The structure of the conventional offline three-dimensional liquid chromatography apparatus which combined the ion exchange (DEAE), reverse phase, and normal phase (NP / HILIC) column is shown. 本発明の第１の実施形態である順相型２次元液体クロマトグラフィ装置の応用例である。It is an application example of the normal phase type two-dimensional liquid chromatography apparatus which is the 1st Embodiment of this invention. ヒト血清中の２−アミノピリジン（ＰＡ）誘導体化糖鎖を本発明の方法で分離分析したクロマトグラムである。2 is a chromatogram obtained by separating and analyzing 2-aminopyridine (PA) derivatized sugar chains in human serum by the method of the present invention. 本発明の第２の実施形態である順相型２次元液体クロマトグラフィ装置の構成と示す。It shows a configuration of a normal phase type two-dimensional liquid chromatography apparatus according to a second embodiment of the present invention.

符号の説明Explanation of symbols

Ａ…水溶液、Ｂ…有機溶媒溶液、ＡＳ…試料注入装置、ＩＥＸ…イオン交換カラム。   A ... aqueous solution, B ... organic solvent solution, AS ... sample injection device, IEX ... ion exchange column.

Claims (7)

複数の溶液を混合し送液するポンプと、試料注入手段と、イオン交換による前段分離カラムと（順相／ヒリック）による後段分離カラムと、分離された成分を検出する一つまたは複数の検出手段から構成される液体クロマトグラフ装置であって、
前記ポンプは、前記試料注入手段に直結され、且つ前記試料注入手段はイオン交換による前記前段分離カラムに直結され、水溶液と有機溶媒溶液の混合液を前記イオン交換による前段分離カラムに送液し、さらに前記混合液の当該有機溶媒溶液の組成比を下げて送液し、さらに前記イオン交換カラムに保持された成分を溶出する水溶液を送液することを特徴とする２次元液体クロマトグラフ装置。 Pump for mixing and feeding a plurality of solutions, sample injection means, pre-separation column by ion exchange, post-separation column by (normal phase / hillick), and one or more detection means for detecting separated components A liquid chromatograph apparatus comprising:
The pump is directly connected to the sample injection means, and the sample injection means is directly connected to the preceding separation column by ion exchange, and sends a mixed solution of an aqueous solution and an organic solvent solution to the preceding separation column by ion exchange , Further, the two-dimensional liquid chromatograph apparatus is characterized in that the composition of the organic solvent solution in the mixed liquid is lowered and fed, and an aqueous solution eluting the components held in the ion exchange column is fed. 請求項１に記載の２次元液体クロマトグラフ装置において、
前記ポンプは、前記イオン交換カラムに保持された成分を溶出する水溶液を送液した後、再度水溶液と有機溶媒溶液の混合液を送液し、さらに前記混合液の当該有機溶媒溶液の組成比を下げて送液し、さらに前記イオン交換カラムに保持された成分を溶出する水溶液を送液することを特徴とする２次元液体クロマトグラフ装置。 The two-dimensional liquid chromatograph apparatus according to claim 1,
The pump sends an aqueous solution that elutes the components held in the ion exchange column, then sends the mixed solution of the aqueous solution and the organic solvent solution again, and further determines the composition ratio of the organic solvent solution in the mixed solution. A two-dimensional liquid chromatograph apparatus, wherein the two-dimensional liquid chromatograph is configured to send an aqueous solution that lowers and sends the solution and further elutes the components held in the ion exchange column. 前記後段分離カラムは、親水性相互作用を持つものである特徴とする請求項１に記載の２次元液体クロマトグラフ装置。 The two-dimensional liquid chromatograph according to claim 1, wherein the latter separation column has a hydrophilic interaction. 請求項１に記載の２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオン、または陰イオン交換カラムであることを特徴とする２次元液体クロマトグラフ装置。 In 2-dimensional liquid chromatography apparatus according to claim 1, 2-dimensional liquid chromatograph and wherein the front separation column is a cation or an anion exchange column. 請求項１に記載の２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオンと陰イオン交換充填剤を１本のカラムに充填したものであることを特徴とする２次元液体クロマトグラフ装置。 In 2-dimensional liquid chromatography apparatus according to claim 1, preceding separation column 2-dimensional liquid chromatography apparatus, characterized in that packed with the cation and anion exchange packing material in one column. 請求項１に記載の２次元液体クロマトグラフィ装置において、前段分離カラムが陽イオンと陰イオン交換充填剤を１本のカラムに充填し、陽（陰）イオンと陰（陽）イオン交換カラムを直列に接続したものであることを特徴とする２次元液体クロマトグラフ装置。 2. The two-dimensional liquid chromatography apparatus according to claim 1, wherein the first separation column is packed with a cation and an anion exchange packing material in one column, and the cation (anion) and anion (cation) ion exchange column are connected in series. A two-dimensional liquid chromatograph apparatus characterized by being connected. 請求項１に記載の２次元液体クロマトグラフ装置において、前記検出手段として質量分析計を用いることを特徴とする２次元液体クロマトグラフ装置。 In a two-dimensional liquid chromatograph apparatus according to claim 1, wherein the detecting means 2-dimensional liquid chromatographic apparatus, which comprises using a mass spectrometer as.
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