JPH09127090A - Method for analyzing sugars - Google Patents
Method for analyzing sugarsInfo
- Publication number
- JPH09127090A JPH09127090A JP7285970A JP28597095A JPH09127090A JP H09127090 A JPH09127090 A JP H09127090A JP 7285970 A JP7285970 A JP 7285970A JP 28597095 A JP28597095 A JP 28597095A JP H09127090 A JPH09127090 A JP H09127090A
- Authority
- JP
- Japan
- Prior art keywords
- liquid chromatography
- filler
- packing material
- column
- analyzing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は液体クロマトグラフ
ィー法による糖類の分析方法に関する。TECHNICAL FIELD The present invention relates to a method for analyzing sugars by a liquid chromatography method.
【０００２】[0002]
【従来の技術】糖類の分析において液体クロマトグラフ
ィー分析は汎用されている分析手法の一つである。この
液体クロマトグラフィー分析、例えば、ＨＰＬＣ（高性
能液体クロマトグラフィー）分析は、一般に順相分配系
液体クロマトグラフィーで行われている。順相分配系液
体クロマトグラフィーとは、極性の比較的強い固定相
と、固定相に比べて極性の弱い移動相を用いる液体クロ
マトグラフィーのことであり、固定相用の充填剤として
は順相分配液体クロマトグラフィー用充填剤が使用さ
れ、例えば、官能基として−ＮＨ2 、−ＣＮ、−ＯＨな
どをもつ充填剤が使用される。2. Description of the Related Art Liquid chromatographic analysis is one of the commonly used analytical methods in the analysis of sugars. This liquid chromatography analysis, for example, HPLC (high performance liquid chromatography) analysis is generally performed by normal phase partitioning liquid chromatography. Normal-phase partitioning liquid chromatography is a liquid chromatography that uses a stationary phase with a relatively strong polarity and a mobile phase with a weaker polarity than the stationary phase. A packing material for liquid chromatography is used, for example, a packing material having —NH 2 , —CN, —OH or the like as a functional group.
【０００３】この糖類の分析で得られるクロマトグラム
において、分析時間を短くすると分離が悪くなるという
問題があった。糖類の分析において各ピークの保持時間
の短縮、各ピークの分離性等のクロマトグラムを改良し
たい場合、その方法としては、溶離液種類、測定温度ま
たはカラムサイズの変更など種々あるが、特に充填剤の
改良が最も効果的である。しかし、目的にあった充填剤
を新たに製造し、それを用いてクロマトグラムを改良す
ることは、他の方法に比べると煩雑であり容易なことで
はない。In the chromatogram obtained by the analysis of this saccharide, there is a problem that the separation becomes worse when the analysis time is shortened. When you want to improve the chromatogram of the retention time of each peak and the separability of each peak in the analysis of saccharides, there are various methods such as changing the type of eluent, measuring temperature or column size, but especially packing material. Improvement of is most effective. However, it is complicated and not easy to newly prepare a packing material suitable for the purpose and improve the chromatogram by using the packing material.
【０００４】また、液体クロマトグラフィーの分析手法
を簡便に改良する一方法として、２種類の充填剤を一本
のカラムに充填されてなるカラムを用いた手法が開示さ
れている。例えば、特開平６−２７３４０１号公報に
は、陽イオン交換基を備えた充填剤と陰イオン交換基を
備えた充填剤を一本のカラムに充填し、移動相としてア
ルカリ性溶液および酸性溶液を用い、これら両溶液を切
り替えてカラム内に流通させて各イオン種を別々に溶出
させて分析を行う、イオン交換液体クロマトグラフィー
による陽イオンおよび陰イオンの分析方法が開示されて
いるが、これは、カラムを交換することなく両方のイオ
ン種を分析して手法の簡便化を図るものであり、測定対
象試料のクロマトグラムの改良はなされていない。ま
た、これは、イオン交換系液体クロマトグラフィーに関
するものであり、順相分配系液体クロマトグラフィーで
はなく、糖類の分析には適用され得ない。Further, as one method for simply improving the analysis method of liquid chromatography, a method using a column in which two kinds of packing materials are packed in one column is disclosed. For example, in JP-A-6-273401, a column having a packing material having a cation exchange group and a packing material having an anion exchange group is packed in one column, and an alkaline solution and an acidic solution are used as a mobile phase. , A method of analyzing cations and anions by ion exchange liquid chromatography, in which both of these solutions are switched and passed through a column and each ionic species is separately eluted for analysis, which is disclosed. It aims at simplifying the method by analyzing both ion species without exchanging the column, and the chromatogram of the sample to be measured has not been improved. It also relates to ion-exchange liquid chromatography, not normal phase partition liquid chromatography, and cannot be applied to the analysis of sugars.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、上記問題点
に鑑みてなされたものであり、その目的は、糖類を短時
間で分離性良く分析し得る液体クロマトグラフィー法に
よる分析方法を提供することにある。SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object thereof is to provide an analysis method by liquid chromatography capable of analyzing saccharides in a short time with good separability. Especially.
【０００６】[0006]
【課題を解決するための手段】請求項１記載の糖類の分
析方法（以下、請求項１記載の発明を本発明１という）
は、糖類を含有する試料を、順相分配液体クロマトグラ
フィー用充填剤（Ａ）と、上記充填剤（Ａ）とは異な
り、かつ上記充填剤（Ａ）の上記糖類に対するクロマト
グラムを改良し得る充填剤（Ｂ）とが一本のカラムに充
填されてなる液体クロマトグラフィー用カラムに導入
し、液体クロマトグラフィー分析を行うことを特徴とす
る。A method for analyzing saccharides according to claim 1 (hereinafter, the invention according to claim 1 is referred to as present invention 1).
Is different from the packing material (A) for normal phase partitioning liquid chromatography and the packing material (A) for a sample containing saccharides, and can improve the chromatogram of the packing material (A) for the saccharides. It is characterized in that the packing material (B) is introduced into a column for liquid chromatography, which is packed in one column, and liquid chromatography analysis is carried out.
【０００７】本発明１でいうクロマトグラムの改良と
は、例えば、各ピークの保持時間の短縮、各ピークの分
離性、ピークのシャープさなどを言う。The improvement of the chromatogram referred to in the present invention 1 means, for example, shortening of retention time of each peak, separability of each peak, sharpness of the peak and the like.
【０００８】本発明１で使用される充填剤（Ａ）は、糖
類の分離に主に作用する充填剤であり、順相分配液体ク
ロマトグラフィー用充填剤に限定される。The packing material (A) used in the present invention 1 is a packing material which mainly acts on the separation of sugars and is limited to packing materials for normal phase partitioning liquid chromatography.
【０００９】上記充填剤（Ａ）としては、順相分配液体
クロマトグラフィー用充填剤であれば特に制限はなく、
公知のいずれもが使用可能である。順相分配液体クロマ
トグラフィー用充填剤とは、前述の順相分配系液体クロ
マトグラフィーに使用される充填剤のことであり、例え
ば、官能基として−ＮＨ2 、−ＣＮ、−ＯＨなどをもつ
充填剤である。上記充填剤（Ａ）の骨格の素材として
も、特に限定されず、有機系、無機系のいずれも使用可
能であり、有機系のものとしては、合成系、天然系のい
ずれでもよい。上記合成系の素材としては、例えば、ア
クリル系、スチレン系の高分子が挙げられ、上記天然系
の素材としては、例えば、セルロース系、キトサン系、
ポリアミノ酸などが挙げられ、更に無機系のものとして
は、例えば、シリカ、ジルコニア等が挙げられる。The filler (A) is not particularly limited as long as it is a filler for normal phase partitioning liquid chromatography.
Any known method can be used. The packing material for normal phase partitioning liquid chromatography is a packing material used in the above-mentioned normal phase partitioning liquid chromatography, and for example, packing material having —NH 2 , —CN, —OH or the like as a functional group. It is an agent. The material of the skeleton of the filler (A) is not particularly limited, either organic type or inorganic type can be used, and the organic type may be either synthetic type or natural type. Examples of the synthetic material include acrylic-based and styrene-based polymers, and examples of the natural material include cellulose-based and chitosan-based materials.
Examples thereof include polyamino acids, and examples of the inorganic type include silica and zirconia.
【００１０】本発明１で使用される充填剤（Ｂ）は、上
記充填剤（Ａ）とは異なり、かつ上記充填剤（Ａ）の糖
類に対するクロマトグラムを改良し得る充填剤に限定さ
れる。上記充填剤（Ｂ）の選択範囲としては、上記のよ
うに限定された範囲内のものであれば、従来から液体ク
ロマトグラフィー用充填剤として使用されてきたいずれ
の充填剤も使用可能であり、具体的には、順相分配液体
クロマトグラフィー用充填剤、イオン交換液体クロマト
グラフィー用充填剤、逆相分配液体クロマトグラフィー
用充填剤、その他（アパタイトや官能基なしなど）が挙
げられる。また、上記充填剤（Ｂ）の骨格の素材として
も、特に限定されず、例えば、前述の充填剤（Ａ）の骨
格の素材として例示したものが例示される。また、充填
剤（Ｂ）は、２種以上混合されて使用されてもよい。The packing material (B) used in the present invention 1 is limited to the packing material which is different from the packing material (A) and can improve the chromatogram of the packing material (A) for sugars. As the selection range of the filler (B), any filler conventionally used as a filler for liquid chromatography can be used as long as it is within the limited range as described above. Specific examples include packing materials for normal phase partitioning liquid chromatography, packing materials for ion exchange liquid chromatography, packing materials for reverse phase partitioning liquid chromatography, and others (without apatite or functional groups). Further, the material of the skeleton of the filler (B) is not particularly limited, and examples thereof include those exemplified as the material of the skeleton of the filler (A). Further, the filler (B) may be used as a mixture of two or more kinds.
【００１１】充填剤（Ａ）と充填剤（Ｂ）の組合せとし
ては、上記充填剤（Ｂ）が順相分配液体クロマトグラフ
ィー用充填剤である場合としては、充填剤（Ａ）と充填
剤（Ｂ）の官能基同士が異なる例としては、例えば、Ｎ
Ｈ2 基をもつ充填剤とＣＮ基をもつ充填剤の組合せ、充
填剤（Ａ）と充填剤（Ｂ）の骨格素材が異なる例として
は、ＮＨ2 基をもつシリカ系素材からなる充填剤とＮＨ
2 基をもつポリマー系素材からなる充填剤の組合せが挙
げられる。また、さらに充填剤（Ａ）と充填剤（Ｂ）の
糖類の分離に関与する官能基が同じであり、骨格素材も
同じであり、ただ充填剤（Ａ）と充填剤（Ｂ）が同一物
でない場合も含まれる。このような場合としては、例え
ば、用いる素材が同一のものでも、その使用量が異なる
場合、或いはポリマー系の素材の場合であって重合条件
（重合温度、時間など）が異なる場合など、処方が異な
れば同一物でなく、このようなものを組み合わせて充填
剤（Ａ）のクロマトグラムを充填剤（Ｂ）によって改良
し得る場合である。As a combination of the filler (A) and the filler (B), when the filler (B) is a filler for normal phase partition liquid chromatography, the filler (A) and the filler ( Examples of different functional groups of B) include, for example, N
An example of a combination of a filler having an H 2 group and a filler having a CN group and different skeleton materials of the filler (A) and the filler (B) is a filler made of a silica-based material having an NH 2 group. NH
An example is a combination of fillers made of a polymer material having two groups. Further, the filler (A) and the filler (B) have the same functional group involved in the separation of sugars, the skeleton materials are also the same, and the filler (A) and the filler (B) are the same. If not, it is included. In such a case, for example, even if the same material is used, the amount used is different, or in the case of a polymer material and the polymerization conditions (polymerization temperature, time, etc.) are different, the formulation is different. If they are different, they are not the same, and a combination of such substances can improve the chromatogram of the packing material (A) with the packing material (B).
【００１２】充填剤（Ｂ）が順相分配液体クロマトグラ
フィー用充填剤以外である場合としては、イオン交換液
体クロマトグラフィー用充填剤、逆相分配液体クロマト
グラフィー用充填剤又はアパタイトが好ましい。上記イ
オン交換液体クロマトグラフィー用充填剤としては、カ
チオン交換基又はアニオン交換基をもつ充填剤が選ば
れ、カチオン交換基をもつものとしては、例えば、官能
基として−ＣＯＯＨ、−ＳＯ3 Ｈなどをもつ充填剤が挙
げられ、アニオン交換基をもつものとしては、例えば、
官能基として−ＮＲ2 、−ＮＲ3 （Ｒは水素原子又はア
ルキル基）などをもつ充填剤が挙げられる。また、上記
逆相分配液体クロマトグラフィー用充填剤としては、例
えば、官能基としてオクチル基、オクタデシル基のよう
な直鎖アルキル基をもつものや、フェニル基などをもつ
充填剤が挙げられる。When the packing material (B) is other than the packing material for normal phase partitioning liquid chromatography, the packing material for ion exchange liquid chromatography, the packing material for reverse phase partitioning liquid chromatography or apatite is preferable. As the above-mentioned packing material for ion exchange liquid chromatography, a packing material having a cation exchange group or an anion exchange group is selected, and as the packing material having a cation exchange group, for example, —COOH, —SO 3 H or the like as a functional group is selected. Examples of the filler having an anion exchange group include, for example,
-NR 2 as a functional group, -NR 3 (R is a hydrogen atom or an alkyl group) filler with the like. Examples of the packing material for reversed phase partition liquid chromatography include packing materials having a linear alkyl group such as an octyl group and octadecyl group as a functional group, and packing materials having a phenyl group and the like.
【００１３】充填剤（Ａ）と充填剤（Ｂ）の使用量の比
率は、用いる充填剤の種類により異なるが、通常、充填
剤（Ａ）と充填剤（Ｂ）の合計量中の充填剤（Ｂ）の含
有量は、５０重量％以下が好ましい。The ratio of the amount of the filler (A) to the amount of the filler (B) used varies depending on the kind of the filler used, but usually, the filler is contained in the total amount of the filler (A) and the filler (B). The content of (B) is preferably 50% by weight or less.
【００１４】本発明１で用いられるカラムは、従来から
液体クロマトグラフィー用カラムとして用いられている
ものであれば、特に制限されないが、直径１〜２０ｍ
ｍ、長さ２０〜５００ｍｍのステンレス管が好ましい。The column used in the present invention 1 is not particularly limited as long as it has been conventionally used as a column for liquid chromatography, but the diameter is 1 to 20 m.
A stainless steel tube having m and a length of 20 to 500 mm is preferable.
【００１５】本発明１で用いられる液体クロマトグラフ
ィー用カラムを製造するには、一本のカラムに充填剤
（Ａ）と充填剤（Ｂ）を充填すればよい。充填順序とし
ては、充填剤（Ａ）及び充填剤（Ｂ）を混合してから充
填してもよいし、別々に充填（この場合は充填剤が多層
状態でカラム内に存在することになる）してもよい。ま
た、充填方法としては湿式充填法が好ましい。具体的に
は、分析に使用する溶離液等に、カラムサイズに見合っ
た量の充填剤を分散させてパッカーに移し、パッカーの
入口側にポンプ、出口側にカラムを接続し、定流量又は
定圧送液して充填する方法が挙げられる。In order to produce the column for liquid chromatography used in the first aspect of the present invention, one column may be packed with the packing material (A) and the packing material (B). As the packing order, the packing (A) and the packing (B) may be mixed and then packed, or packed separately (in this case, the packing is present in the column in a multilayer state). You may. A wet filling method is preferable as the filling method. Specifically, in the eluent used for analysis, etc., disperse an amount of packing material suitable for the column size and transfer to a packer, connect a pump to the inlet side of the packer and a column to the outlet side, and set a constant flow rate or constant flow rate. A method of filling by pressure-feeding the liquid may be mentioned.
【００１６】本発明１で分析される糖類を含有する試料
としては、糖を含む化合物を含有する試料であれば特に
限定されず、糖を含む化合物としては、例えば、単糖
類、２〜２０糖類のオリゴ糖類、糖アルコール類などが
挙げられる。The sample containing a saccharide analyzed in the present invention 1 is not particularly limited as long as it contains a compound containing a sugar. Examples of the compound containing a sugar include monosaccharides and 2 to 20 saccharides. The oligosaccharides, sugar alcohols, etc.
【００１７】本発明１の糖類の分析方法は、分析に使用
するカラムとして、上述の液体クロマトグラフィー用カ
ラムを使用することの他は、従来の液体クロマトグラフ
ィー法による糖類の分析方法と同様である。以下、本発
明１の糖類の分析方法について詳述する。The method for analyzing saccharides of the present invention 1 is the same as the method for analyzing sugars by the conventional liquid chromatography method except that the above-mentioned column for liquid chromatography is used as a column used for the analysis. . Hereinafter, the method for analyzing a saccharide of the present invention 1 will be described in detail.
【００１８】図１は、上述の液体クロマトグラフィー用
カラムを使用して糖類の分析をする際の装置の構成の一
例である。液体クロマトグラフィー用カラム１に移動相
２を送液ポンプ３により供給しながら、サンプラー４か
ら糖類を含有する試料を導入する。液体クロマトグラフ
ィー用カラム１によって分離された試料は、検出器５よ
って検出され、その結果が記録計６に記録される。な
お、糖類を含有する試料に必要に応じて公知の前処理が
施されてもよい。FIG. 1 shows an example of the constitution of an apparatus for analyzing saccharides using the above-mentioned liquid chromatography column. While supplying the mobile phase 2 to the liquid chromatography column 1 by the liquid feed pump 3, the sample containing the saccharide is introduced from the sampler 4. The sample separated by the liquid chromatography column 1 is detected by the detector 5, and the result is recorded in the recorder 6. The sample containing saccharides may be subjected to a known pretreatment, if necessary.
【００１９】上記移動相としては、順相分配モードにお
ける公知の溶離液が用いられる。例えば、水系では、無
機酸（リン酸など）、有機酸（クエン酸など）および／
またはこれらの塩よりなる緩衝液などが用いられる。ま
た、有機溶媒系では、例えば、メタノール、エタノー
ル、アセトニトリルなどが用いられる。また、特に、水
と有機溶媒の混合系が好ましく用いられる。移動相の通
液する速度（流速）は、一般に０．５〜５ｍｌ／ｍｉｎ
程度が選ばれる。As the mobile phase, a known eluent in the normal phase distribution mode is used. For example, in aqueous systems, inorganic acids (such as phosphoric acid), organic acids (such as citric acid) and / or
Alternatively, a buffer containing these salts is used. Further, in the organic solvent system, for example, methanol, ethanol, acetonitrile or the like is used. In particular, a mixed system of water and an organic solvent is preferably used. The speed (flow rate) of passing the mobile phase is generally 0.5 to 5 ml / min.
The degree is chosen.
【００２０】上記の分離については、公知の方法が採用
され、適当濃度の移動相により試料が分離溶出される。
また、クロマトグラムの改良ため、分析途中で溶離液濃
度や流速等を変えることもできる。また、液体クロマト
グラフィー用カラム等を恒温槽内に設置することもでき
る。For the above separation, a known method is adopted, and the sample is separated and eluted with a mobile phase having an appropriate concentration.
Further, in order to improve the chromatogram, it is possible to change the concentration of the eluent or the flow rate during the analysis. Further, a liquid chromatography column or the like can be installed in a constant temperature bath.
【００２１】上記の検出についても公知の技術が用いら
れ、例えば、紫外可視分光光度計、示差屈折計などが好
適に使用される。Known techniques are also used for the above detection, and for example, an ultraviolet-visible spectrophotometer and a differential refractometer are preferably used.
【００２２】[0022]
【発明の実施の形態】以下、本発明の実施例を説明す
る。実施例における分析装置は、前記図１のように構成
して行った。なお、各装置は、以下のものを使用した。 送液ポンプ３：島津製作所社製、ＬＣ−９Ａ。 サンプラー４：オートサンプラー。積水化学工業社製、
ＡＳＵ−４２０。 検出器５：示差屈折計。昭和電工社製、ＲＩ ＳＥ−５
１。Embodiments of the present invention will be described below. The analysis apparatus in the examples was constructed as shown in FIG. The following devices were used for each device. Liquid sending pump 3: LC-9A manufactured by Shimadzu Corporation. Sampler 4: Auto sampler. Sekisui Chemical Co., Ltd.
ASU-420. Detector 5: differential refractometer. Showa Denko KK, RI SE-5
One.
【００２３】実施例において使用した充填剤は、以下の
通りである。 充填剤 順相−１ 市販の順相分配液体クロマトグラフィー用充填剤（μＢ
ｏｎｄａｓｐｈｅｒｅ−ＮＨ2 、Ｗａｔｅｒｓ社製）。
骨格の素材として、シリカが使用され、官能基として−
ＮＨ2 を有している。The fillers used in the examples are as follows. Packing Agent Normal Phase-1 Commercially available packing material for normal phase partitioning liquid chromatography (μB
ondasphere-NH 2 , manufactured by Waters).
Silica is used as the material of the skeleton, and as the functional group-
It has NH 2 .
【００２４】充填剤 順相−２ 以下のように、特開平３−１８８９４２号公報の方法に
より順相分配液体クロマトグラフィー用充填剤を製造し
た。アリルアミン（和光純薬社製）２４ｇ、トリエチレ
ングリコールジメタクリレート（新中村化学社製）４０
０ｇ及びｔ−ブチルパーオキシネオデカノエート（重合
開始剤、和光純薬社製）１．５ｇを混合・溶解し、２０
００ｍｌの４重量％ポリビニルアルコール（日本合成社
製）水溶液に添加した。これに、リン酸７８ｇを添加
し、その後、窒素置換下で攪拌しながら５０℃で２４時
間重合し、生成物を熱水、０．１Ｎ水酸化ナトリウム水
溶液及びアセトンで順次、洗浄し、乾燥した。得られた
粒子を空気分級機（日鉄鉱業社製）で分級して６〜８μ
ｍの粒子を集め、これを充填剤順相−２とした。充填剤
順相−２は、官能基として−ＮＨ 2 を有する。Filler Normal Phase-2 As described below, according to the method of JP-A-3-188942.
To produce packing materials for normal phase partition liquid chromatography
Was. Allylamine (Wako Pure Chemical Industries, Ltd.) 24g, Triethyl
Glycol dimethacrylate (manufactured by Shin Nakamura Chemical Co., Ltd.) 40
0 g and t-butyl peroxy neodecanoate (polymerized
Initiator, manufactured by Wako Pure Chemical Industries, Ltd.) 1.5 g are mixed and dissolved, and 20
00 ml of 4 wt% polyvinyl alcohol (Nippon Gosei Co., Ltd.
Manufactured). Add 78g of phosphoric acid to this
Then, at 24 hours at 50 ° C with stirring under nitrogen replacement.
The product is polymerized for a while, and the product is hot water, 0.1N sodium hydroxide water.
It was washed successively with the solution and acetone and dried. Got
Particles are classified with an air classifier (manufactured by Nittetsu Mining Co., Ltd.) to 6 to 8μ.
m particles were collected and designated as filler normal phase-2. filler
Normal phase-2 is -NH as a functional group. TwoHaving.
【００２５】充填剤 カチオン−１ 以下のように、特開平３−７３８４８号公報の方法によ
りカチオン交換充填剤を製造した。ジエチレングリコー
ルジメタクリレート（新中村化学社製）２００ｇにベン
ゾイルパーオキサイド（重合開始剤、和光純薬社製）１
ｇを溶解し、３重量％ポリビニルアルコール（日本合成
社製）水溶液に添加した。攪拌しながら窒素を導入し、
８０℃で２時間重合した。室温まで冷却した後、アクリ
ル酸４０ｇを添加して再び８０℃に加熱して２時間重合
した。得られた粒子を水およびアセトンで順次、洗浄
し、乾燥した後、空気分級機（日鉄鉱業社製）で分級し
て６〜８μｍの粒子を集め、これを充填剤カチオン−１
とした。充填剤カチオン−１は、官能基として−ＣＯＯ
Ｈを有する。Filler Cation-1 A cation exchange filler was produced by the method described in JP-A-3-73848 as follows. To 200 g of diethylene glycol dimethacrylate (manufactured by Shin Nakamura Chemical Co., Ltd.), 1 benzoyl peroxide (polymerization initiator, manufactured by Wako Pure Chemical Industries, Ltd.)
g was dissolved and added to a 3 wt% polyvinyl alcohol (Nippon Gosei) aqueous solution. Introduce nitrogen while stirring,
Polymerization was carried out at 80 ° C. for 2 hours. After cooling to room temperature, 40 g of acrylic acid was added, and the mixture was heated again to 80 ° C. and polymerized for 2 hours. The obtained particles are washed successively with water and acetone, dried and then classified with an air classifier (manufactured by Nittetsu Mining Co., Ltd.) to collect particles of 6 to 8 μm, which are used as filler cation-1.
And The filler cation-1 has -COO as a functional group.
H.
【００２６】充填剤 逆相−１ 市販の逆相分配液体クロマトグラフィー用充填剤（Ｍｉ
ｃｒｏｎｅｘ ＲＰ−３０、積水化学工業社製）。骨格
の素材として、スチレン系ポリマーが使用され、官能基
としてフェニル基を有している。Packing material Reversed phase-1 A commercially available packing material for reversed phase partition liquid chromatography (Mi.
cronex RP-30, manufactured by Sekisui Chemical Co., Ltd.). A styrene-based polymer is used as a material for the skeleton and has a phenyl group as a functional group.
【００２７】（実施例１〜３、比較例１） 液体クロマトグラフィー用カラムの製造 表１に示した充填剤（Ａ）と充填剤（Ｂ）を、表１に示
した使用量（ｇ）で３０ｍｌの５０重量％メタノール水
溶液に分散した（比較例１は、充填剤（Ａ）のみ使
用）。内径６ｍｍ、長さ５０ｍｍのステンレス製カラム
を接続したパッカー（容量３０ｍｌ：梅谷精機社製）に
上記分散液を加え、送液ポンプ（島津製作所社製、ＬＣ
−６ＡＤ）をパッカーに接続して、定圧充填を行ってカ
ラムを製造した。(Examples 1 to 3, Comparative Example 1) Production of Liquid Chromatography Column The packing material (A) and the packing material (B) shown in Table 1 were used in the amounts (g) shown in Table 1. It was dispersed in 30 ml of a 50 wt% methanol aqueous solution (Comparative Example 1 used only the filler (A)). The dispersion liquid was added to a packer (capacity 30 ml: Umeya Seiki Co., Ltd.) connected to a stainless steel column having an inner diameter of 6 mm and a length of 50 mm, and a liquid feed pump (Shimadzu Corporation, LC).
-6AD) was connected to a packer and constant pressure packing was performed to manufacture a column.
【００２８】[0028]
【表１】 [Table 1]
【００２９】糖類の分離 上記装置に上記液体クロマトグラフィー用カラムを取り
つけた装置で、糖類を含有する試料として糖類標品（フ
ルクトース、グルコース、シュクロース、マルトース及
びラクトースを含有）を水に溶解したものを分析した。
溶出には、アセトニトリル８容と水２容の混合溶液を使
用し、流速は２．０ｍｌ／ｍｉｎとした。実施例１で得
られたクロマトグラムを図２に、比較例１で得られたク
ロマトグラムを図３に示した。なお、実施例２、３のク
ロマトグラムも実施例１のクロマトグラムとほぼ同様で
あった。なお、図２及び３における各ピークは、１０フ
ルクトース、１１グルコース、１２シュクロース、１３
マルトース、１４ラクトースである。Separation of saccharides An apparatus in which the above-mentioned liquid chromatography column is attached to the above apparatus, in which a saccharide standard (containing fructose, glucose, sucrose, maltose and lactose) is dissolved in water as a sample containing saccharides. Was analyzed.
For elution, a mixed solution of 8 volumes of acetonitrile and 2 volumes of water was used, and the flow rate was 2.0 ml / min. The chromatogram obtained in Example 1 is shown in FIG. 2, and the chromatogram obtained in Comparative Example 1 is shown in FIG. The chromatograms of Examples 2 and 3 were almost the same as those of Example 1. Each peak in FIGS. 2 and 3 represents 10 fructose, 11 glucose, 12 sucrose, 13
Maltose and 14 lactose.
【００３０】[0030]
【発明の効果】本発明１〜３の糖類の分析方法の構成は
上記の通りであり、糖類のクロマトグラムの改良がなさ
れ得、糖類を短時間で分離性良く分析し得る。また、本
発明１〜３の糖類の分析方法に使用される液体クロマト
グラフィー用カラムは、順相分配液体クロマトグラフィ
ー用充填剤（Ａ）と、上記充填剤（Ａ）とは異なり、か
つ上記充填剤（Ａ）の上記糖類に対するクロマトグラム
を改良し得る充填剤（Ｂ）とが一本のカラムに充填され
てなる液体クロマトグラフィー用カラムであり、その製
造が容易である。従って、本発明１〜３の糖類の分析方
法は、簡便な方法でより正確に糖類を分析できる方法で
ある。The constitution of the method for analyzing saccharides of the present invention 1 to 3 is as described above, the chromatogram of saccharides can be improved, and saccharides can be analyzed in a short time with good separability. Moreover, the column for liquid chromatography used in the method for analyzing saccharides of the present inventions 1 to 3 is different from the packing material (A) for normal phase partitioning liquid chromatography and the packing material (A), and the packing material is the above packing. This is a column for liquid chromatography in which one column is packed with a packing material (B) capable of improving the chromatogram of the agent (A) with respect to the above saccharides, and its production is easy. Therefore, the method for analyzing saccharides of the present inventions 1 to 3 is a method that can analyze sugars more accurately by a simple method.
【００３１】[0031]
【図１】本発明の糖類の分析を行う際の装置の構成の一
例である。FIG. 1 is an example of the configuration of an apparatus for analyzing a saccharide of the present invention.
【図２】実施例１のクロマトグラムである。FIG. 2 is a chromatogram of Example 1.
【図３】比較例１のクロマトグラムである。FIG. 3 is a chromatogram of Comparative Example 1.
１ 液体クロマトグラフィー用カラム ２ 移動相 ３ 送液ポンプ ４ サンプラー ５ 検出器 ６ 記録計 1 Column for liquid chromatography 2 Mobile phase 3 Liquid feed pump 4 Sampler 5 Detector 6 Recorder
Claims (3)
ロマトグラフィー用充填剤（Ａ）と、上記充填剤（Ａ）
とは異なり、かつ上記充填剤（Ａ）の上記糖類に対する
クロマトグラムを改良し得る充填剤（Ｂ）とが一本のカ
ラムに充填されてなる液体クロマトグラフィー用カラム
に導入し、液体クロマトグラフィー分析を行うことを特
徴とする糖類の分析方法。1. A packing material (A) for normal phase partitioning liquid chromatography is applied to a sample containing a saccharide, and the packing material (A).
And a packing material (B) capable of improving the chromatogram of the packing material (A) with respect to the saccharides, which is packed in one column and introduced into a column for liquid chromatography, and liquid chromatography analysis A method for analyzing sugars, which comprises:
トグラフィー用充填剤である請求項１記載の糖類の分析
方法。2. The method for analyzing saccharides according to claim 1, wherein the packing material (B) is a packing material for normal phase partition liquid chromatography.
マトグラフィー用充填剤、逆相分配液体クロマトグラフ
ィー用充填剤又はアパタイトである請求項１記載の糖類
の分析方法。3. The method for analyzing saccharides according to claim 1, wherein the packing material (B) is a packing material for ion exchange liquid chromatography, a packing material for reversed phase partition liquid chromatography, or apatite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28597095A JP3561351B2 (en) | 1995-11-02 | 1995-11-02 | Sugar analysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28597095A JP3561351B2 (en) | 1995-11-02 | 1995-11-02 | Sugar analysis method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09127090A true JPH09127090A (en) | 1997-05-16 |
| JP3561351B2 JP3561351B2 (en) | 2004-09-02 |
Family
ID=17698332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28597095A Expired - Fee Related JP3561351B2 (en) | 1995-11-02 | 1995-11-02 | Sugar analysis method |
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| Country | Link |
|---|---|
| JP (1) | JP3561351B2 (en) |
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| WO2002027038A1 (en) * | 2000-09-29 | 2002-04-04 | Danisco Sweeteners Oy. | Use of a weakly acid cation exchange resin for chromatographic separation of carbohydrates |
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