JP2938632B2 - Chromatographic separation method - Google Patents
Chromatographic separation methodInfo
- Publication number
- JP2938632B2 JP2938632B2 JP3245681A JP24568191A JP2938632B2 JP 2938632 B2 JP2938632 B2 JP 2938632B2 JP 3245681 A JP3245681 A JP 3245681A JP 24568191 A JP24568191 A JP 24568191A JP 2938632 B2 JP2938632 B2 JP 2938632B2
- Authority
- JP
- Japan
- Prior art keywords
- water
- salt
- separation
- mobile phase
- organic solvent
- 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.)
- Expired - Fee Related
Links
Landscapes
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、液体クロマトグラフィ
ーにより化合物と化合物を分離する方法に関するもので
あり、研究・製造管理などにおける分析及び分離精製に
用いることができる。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating compounds from each other by liquid chromatography, and can be used for analysis and separation and purification in research and production control.
【０００２】[0002]
【従来の技術及び発明が解決しようとする課題】多糖誘
導体、あるいはこれをシリカゲルなどの担体に担持した
分離剤は、液体クロマトグラフィー用固定相として光学
異性体をはじめとする様々の構造的に類似した化合物
（ジアステレオマーなどの立体異性体、位置異性体、幾
何異性体、不飽和度の異なる化合物、ホモローグに属す
る化合物など）の分離において、優れた特性を示すこと
が明らかになっている（第28回HPLC研究談話会要旨集、
ｐ25、26引用）。この種の固定相を利用する場合、従来
は実質的に順相系の移動相（具体的には、ヘキサンなど
の非極性有機溶剤とアルコール類の混液）のみを用いて
きた。2. Description of the Related Art Polysaccharide derivatives, or separating agents in which they are supported on a carrier such as silica gel, are used as stationary phases for liquid chromatography in various structurally similar forms including optical isomers. It has been shown that the compound exhibits excellent properties in the separation of compounds (stereoisomers such as diastereomers, positional isomers, geometric isomers, compounds having different degrees of unsaturation, compounds belonging to homologues, etc.) ( 28th HPLC Research Discourse Abstracts,
p. 25, 26). In the case of using such a stationary phase, conventionally, only a normal phase mobile phase (specifically, a mixed solution of a nonpolar organic solvent such as hexane and an alcohol) has been used.
【０００３】最近になって水溶性有機溶剤と、水または
緩衝液の混液が実用上有用であることが発見された（特
開平３−27326 号公報）が、ここでは水溶性有機溶剤
（好ましくはアセトニトリルが挙げられるが、他、公知
の水溶性有機溶剤）と、水、または各種塩類を含む緩衝
液（例えば、リン酸、酢酸、過塩素酸緩衝液）の10：１
〜１：10の混合比のものが、移動相として有用であると
述べられている。実際には、水溶性有機溶剤と水の混液
では、アミンやカルボン酸を中心として分離しにくい化
合物が多く、酸性緩衝液と有機溶剤の混合液を用いるこ
とによって良好な分離を得ている例が示されている。し
かしながら、緩衝液を調製するためには二種の物質を一
定量ずつ溶解することが必要であるため、操作が煩雑で
あり、また、酸性緩衝液中では、しばしば分解する試料
があるなどの問題があった。Recently, it has been discovered that a mixture of a water-soluble organic solvent and water or a buffer solution is practically useful (Japanese Patent Laid-Open No. 27326/1991). Acetonitrile may be mentioned, and other known water-soluble organic solvents) and water or a buffer containing various salts (eg, phosphate, acetic acid, perchlorate buffer) at a ratio of 10: 1.
Mixture ratios of １ ： 1: 10 are stated to be useful as mobile phases. In fact, in a mixed solution of a water-soluble organic solvent and water, there are many compounds that are difficult to separate, mainly amines and carboxylic acids, and good separation is obtained by using a mixed solution of an acidic buffer and an organic solvent. It is shown. However, the preparation of the buffer requires the dissolution of two substances in a fixed amount, which makes the operation complicated, and in acidic buffers, there are samples that often decompose. was there.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上に述べ
た従来の溶離液系の問題点を解決するため鋭意検討を行
い、その結果、単なる塩の水溶液、あるいは、これに水
溶性有機溶剤を混合した溶液、あるいは、水溶性有機溶
剤に直接塩のみを溶解した溶液、言い換えるならば、水
と水溶性有機溶剤の 100：０〜０：100 の混液に、単に
塩類を添加した溶液が、簡単に調製でき、しかも、中性
に近い条件で良い分離を与えることを見出し、本発明を
完成するに至った。即ち、本発明は、多糖誘導体を有効
成分とする分離剤を用いて、液体クロマトグラフィーを
行うとき、移動相として水と水溶性有機溶剤の 100：０
〜０：100の混液に、過塩素酸塩、チオシアン酸塩、硝
酸塩、ヨウ化物、臭化物、塩化物、パーフルオロアルカ
ン酸、グアニジニウム塩、第四級アンモニウム塩、ルビ
ジウム塩、カリウム塩、セシウム塩から選ばれるカオト
ロピック性が強い塩類を添加した液を用いることを特徴
とする各種化合物の液体クロマトグラフィーによる分離
法を提供するものである。DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies in order to solve the above-mentioned problems of the conventional eluent system. A solution obtained by mixing an organic solvent or a solution in which only a salt is directly dissolved in a water-soluble organic solvent, in other words, a solution obtained by simply adding salts to a mixture of water and a water-soluble organic solvent in a ratio of 100: 0 to 0: 100. However, they have found that they can be easily prepared and give good separation under nearly neutral conditions, and have completed the present invention. That is, according to the present invention, when liquid chromatography is performed using a separating agent containing a polysaccharide derivative as an active ingredient, 100: 0 of water and a water-soluble organic solvent are used as mobile phases.
~ 0: 100 perchlorate, thiocyanate, nitrate
Acid salt, iodide, bromide, chloride, perfluoroalka
Acid, guanidinium salt, quaternary ammonium salt, ruby
Kaoto selected from indium, potassium and cesium salts
An object of the present invention is to provide a method for separating various compounds by liquid chromatography, characterized by using a liquid to which salts having strong tropic properties are added.
【０００５】本発明に用いられる水溶性有機溶剤として
は、水と混和するものであればいかなるものでもよい
が、好ましくは、アセトニトリル、メタノール、エタノ
ールが挙げられるが、他の公知の水溶性有機溶剤も用い
うる。本発明で用いられる塩類は、カオトロピックな性
質が強い（「蛋白質・酵素の基礎実験法」ｐ22, 23, 堀
尾武一, 山下仁平編集, 南江堂より引用）とされている
ものであり、過塩素酸塩、チオシアン酸塩、硝酸塩、ヨ
ウ化物、臭化物、塩化物、パーフルオロアルカン酸、グ
アニジニウム塩、第四級アンモニウム塩、ルビジウム
塩、カリウム塩、セシウム塩から選ばれる。本発明にお
いて、実際に上記の移動相を用いる時には、上記の塩の
一種もしくは何種かを0.001mM から飽和の濃度において
前記の水あるいは水と水溶性有機溶剤との混合液あるい
は水溶性有機溶剤に溶解したものを移動相としてカラム
に送液する。本移動相は、他に保持の強さ、分離の良さ
を調整するための他の成分を含んでいても良い。The water-soluble organic solvent used in the present invention may be any one as long as it is miscible with water, and preferably includes acetonitrile, methanol and ethanol, and other known water-soluble organic solvents. Can also be used. Salts used in the present invention, the chaotropic strong nature ( "Basic Experimental Methods of Proteins and Enzymes" p22, 23, Buichi Horio, Nidaira Yamashita editing, from quotation Nankodo) are those which are with, perchloric acid salt, thiocyanate, nitrate, iodide, bromide, chloride, perfluoro alkanoic acids, guanidinium salts, quaternary ammonium salts, rubidium salts, potassium salts, selected from cesium salt. In the present invention, when the above-mentioned mobile phase is actually used, one or more of the above-mentioned salts are mixed with the above water or a mixture of water and a water-soluble organic solvent or a water-soluble organic solvent at a concentration of 0.001 mM to saturation. The solution dissolved in is sent to the column as a mobile phase. The mobile phase may further contain other components for adjusting the strength of retention and the goodness of separation.
【０００６】本発明に用いられる分離剤は多糖誘導体を
有効成分とするものであり、具体的には、特開昭63−60
944 号公報ｐ363 に開示されているもの、即ち、合成多
糖、天然多糖、天然物変成多糖の誘導体が用いられ、好
ましくは高純度の多糖を容易に得ることのできるセルロ
ース、アミロース、β−1,4 −キトサン、キチン、β−
1,4 −マンナン、β−1,4 −キシラン、イヌリン、α−
1,3 −グルカン、β−1,3 −グルカン等の多糖の有する
水酸基上の水素原子の一部あるいは全部、好ましくは85
％以上を他の原子団で置換したものである。これらの中
で最も有効な分離剤の例としてはセルローストリスフェ
ニルカルバメートを挙げることができる。[0006] The separating agent used in the present invention contains a polysaccharide derivative as an active ingredient.
No. 944, p 363, namely, synthetic polysaccharides, natural polysaccharides and derivatives of modified natural polysaccharides are used, and preferably, cellulose, amylose, β-1, 4--chitosan, chitin, β-
1,4-mannan, β-1,4-xylan, inulin, α-
Part or all of the hydrogen atoms on the hydroxyl groups of polysaccharides such as 1,3-glucan and β-1,3-glucan, preferably 85
% Or more is replaced with another atomic group. The most effective separating agent among these is cellulose trisphenyl carbamate.
【０００７】[0007]
【作用】本発明では、移動相中に単なる各種の塩を添加
することで分離能の向上をもたらしたが、この理由は、
完全に明らかにはなっていない。しかし、岩波理化学辞
典, 第４版（ｐ428), 久保亮五他編, 岩波書店に記され
ているように、前述のカオトロピックな性質の強いとさ
れているイオンは、構造破壊イオンと呼ばれ、溶質周囲
に水分子を水和しにくくする性質のあることが知られて
いる。このため、試料分子の拡散速度や水和に影響を与
え、これが多糖誘導体への吸着に影響を与えていること
が想像される。According to the present invention, the separation ability is improved by simply adding various salts to the mobile phase.
Not completely revealed. However, as described in the Iwanami Scientific and Chemical Dictionary, 4th edition (p. 428), Ryogo Kubo et al., Iwanami Shoten, the aforementioned ions that are considered to have strong chaotropic properties are called structural disruption ions, It is known that there is a property that makes it difficult for water molecules to hydrate around the solute. For this reason, it is supposed that this affects the diffusion rate and hydration of the sample molecule, and this affects the adsorption to the polysaccharide derivative.
【０００８】[0008]
【実施例】以下、本発明を実施例及び比較例によって詳
述するが、本発明はこれらの実施例に限定されるもので
ないことは既に述べた理由より明白である。尚、実施
例、比較例中に用いられるパラメーターk'及びαは以下
のように定義される。EXAMPLES The present invention will be described below in detail with reference to Examples and Comparative Examples, but it is clear from the reasons already described that the present invention is not limited to these Examples. The parameters k ′ and α used in the examples and comparative examples are defined as follows.
【０００９】[0009]
【数１】 (Equation 1)
【００１０】実施例１ 移動相として0.1N−NaClO4水溶液／CH3CN ＝60／40（体
積比）の混合液を用いて、プロプラノールの光学異性体
を分離した。カラムとしては、セルロース・トリス（3,
5 −ジメチルフェニル）カルバメートをシリカゲル上に
担持した固定相を充填した長さ25cm、内径0.46cmの市販
のCHIRALCEL OD（ダイセル化学工業株式会社製）を用い
た。移動相流速は 1.0ml／min、カラム温度は20℃とし
た。溶離する光学異性体の検出は紫外検出器を用い、波
長は254nm とした。分離結果として、クロマトグラムを
図１に示し、両エナンチオマーの保持時間、容量比及び
分離係数を表１に示した。Example 1 An optical isomer of propranolol was separated using a mixture of 0.1N-NaClO 4 aqueous solution / CH 3 CN = 60/40 (volume ratio) as a mobile phase. As the column, cellulose Tris (3,
A commercially available CHIRALCEL OD (manufactured by Daicel Chemical Industries, Ltd.) having a length of 25 cm and an inner diameter of 0.46 cm packed with a stationary phase having 5-dimethylphenyl) carbamate supported on silica gel was used. The mobile phase flow rate was 1.0 ml / min, and the column temperature was 20 ° C. The eluting optical isomer was detected using an ultraviolet detector, and the wavelength was 254 nm. As a result of the separation, a chromatogram is shown in FIG. 1, and a retention time, a volume ratio and a separation coefficient of both enantiomers are shown in Table 1.
【００１１】実施例２ 移動相として0.5N−NaClO4水溶液／CH3CN ＝60／40（体
積比）の混合液を用いて、プロプラノールの光学異性体
を分離した。実験条件は、移動相流速を0.5ml／min 、
カラム温度を25℃とした以外は実施例１と同様にした。
分離結果として、クロマトグラムを図２に示し、両エナ
ンチオマーの保持時間、容量比及び分離係数を表１に示
した。Example 2 Using a mixture of 0.5N-NaClO 4 aqueous solution / CH 3 CN = 60/40 (volume ratio) as a mobile phase, optical isomers of propranol were separated. The experimental conditions were as follows: mobile phase flow rate of 0.5 ml / min,
Example 1 was repeated except that the column temperature was 25 ° C.
As a result of the separation, a chromatogram is shown in FIG. 2, and a retention time, a volume ratio and a separation coefficient of both enantiomers are shown in Table 1.
【００１２】比較例１ 移動相としてpH3.0 0.1N−HClO4 水溶液／CH3CN ＝60／
40（体積比）の混合液を用いて、プロプラノールの光学
異性体を分離した。実験条件は実施例１と同様にした。
分離結果として、クロマトグラムを図３に示し、両エナ
ンチオマーの保持時間、容量比及び分離係数を表１に示
した。COMPARATIVE EXAMPLE 1 pH 3.0 0.1N-HClO 4 aqueous solution / CH 3 CN = 60 /
Using a 40 (volume ratio) mixture, the optical isomers of propranol were separated. The experimental conditions were the same as in Example 1.
As a result of the separation, a chromatogram is shown in FIG. 3, and a retention time, a volume ratio and a separation coefficient of both enantiomers are shown in Table 1.
【００１３】比較例２ 移動相として H2O／CH3CN ＝60／40（体積比）の混合液
を用いて、プロプラノールの光学異性体を分離した。実
験条件は、実施例１と同様にした。分離結果として、ク
ロマトグラムを図４に示し、両エナンチオマーの保持時
間、容量比及び分離係数を表１に示した。Comparative Example 2 Using a mixture of H 2 O / CH 3 CN = 60/40 (volume ratio) as a mobile phase, an optical isomer of propranol was separated. The experimental conditions were the same as in Example 1. As a result of the separation, a chromatogram is shown in FIG. 4, and a retention time, a volume ratio and a separation coefficient of both enantiomers are shown in Table 1.
【００１４】[0014]
【表１】 [Table 1]
【００１５】実施例３ 移動相として0.1N NaClO4 水溶液／CH3CN ＝80／20（体
積比）の混合液を用いて、ホマトロピンの光学異性体を
分離した。実験条件は、移動相流速を0.5 ml／min 、カ
ラム温度を25℃とした以外は実施例１と同様にした。分
離結果として、クロマトグラムを図５に示し、両エナン
チオマーの保持時間、容量比及び分離係数を表２に示し
た。Example 3 An optical isomer of fomatropine was separated using a mixture of 0.1N NaClO 4 aqueous solution / CH 3 CN = 80/20 (volume ratio) as a mobile phase. The experimental conditions were the same as in Example 1 except that the mobile phase flow rate was 0.5 ml / min and the column temperature was 25 ° C. As a result of the separation, a chromatogram is shown in FIG. 5, and a retention time, a volume ratio and a separation coefficient of both enantiomers are shown in Table 2.
【００１６】[0016]
【表２】 [Table 2]
【００１７】[0017]
【発明の効果】本発明の移動相を用いる方法は、該移動
相が水と有機溶剤の混液に単に塩を溶かしただけで、簡
単に調製でき、しかも液性が中性に近い状態で良い分離
を与える。従って、本発明の方法は各種化合物の分析一
般、特に血漿、血清、体液など水を主とする試料液中に
含まれる物質の分析を容易ならしめるものである。According to the method of the present invention using a mobile phase, the mobile phase can be easily prepared by simply dissolving a salt in a mixture of water and an organic solvent, and the liquid phase may be in a state close to neutrality. Give separation. Therefore, the method of the present invention facilitates the analysis of various compounds in general, particularly the analysis of substances contained in water-based sample solutions such as plasma, serum, and body fluid.
【図１】実施例１の分離結果を示すクロマトグラムであ
る。FIG. 1 is a chromatogram showing a separation result of Example 1.
【図２】実施例２の分離結果を示すクロマトグラムであ
る。FIG. 2 is a chromatogram showing a separation result of Example 2.
【図３】比較例１の分離結果を示すクロマトグラムであ
る。FIG. 3 is a chromatogram showing a separation result of Comparative Example 1.
【図４】比較例２の分離結果を示すクロマトグラムであ
る。FIG. 4 is a chromatogram showing a separation result of Comparative Example 2.
【図５】実施例３の分離結果を示すクロマトグラムであ
る。FIG. 5 is a chromatogram showing a separation result of Example 3.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ｃ０７Ｄ 451/10 Ｃ０７Ｄ 451/10 Ｇ０１Ｎ 30/26 Ｇ０１Ｎ 30/26 Ａ 30/88 30/88 Ｗ (58)調査した分野(Int.Cl.6，ＤＢ名) C07B 57/00 C07B 63/00 C07C 217/30 C07D 451/10 G01N 30/26 G01N 30/88 ────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. 6 Identification code FI C07D 451/10 C07D 451/10 G01N 30/26 G01N 30/26 A 30/88 30/88 W (58) Field surveyed (Int. .Cl. 6 , DB name) C07B 57/00 C07B 63/00 C07C 217/30 C07D 451/10 G01N 30/26 G01N 30/88
Claims (1)
いて、液体クロマトグラフィーを行うとき、移動相とし
て水と水溶性有機溶剤の 100：０〜０：100の混液に、
過塩素酸塩、チオシアン酸塩、硝酸塩、ヨウ化物、臭化
物、塩化物、パーフルオロアルカン酸、グアニジニウム
塩、第四級アンモニウム塩、ルビジウム塩、カリウム
塩、セシウム塩から選ばれるカオトロピック性が強い塩
類を添加した液を用いることを特徴とする各種化合物の
液体クロマトグラフィーによる分離法。1. When liquid chromatography is performed using a separating agent containing a polysaccharide derivative as an active ingredient, a liquid mixture of 100: 0 to 0: 100 of water and a water-soluble organic solvent is used as a mobile phase.
Perchlorate, thiocyanate, nitrate, iodide, bromide
Substance, chloride, perfluoroalkanoic acid, guanidinium
Salt, quaternary ammonium salt, rubidium salt, potassium
Strongly chaotropic salt selected from salt and cesium salt
A method for separating various compounds by liquid chromatography, characterized by using a liquid to which compounds are added.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3245681A JP2938632B2 (en) | 1991-09-25 | 1991-09-25 | Chromatographic separation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3245681A JP2938632B2 (en) | 1991-09-25 | 1991-09-25 | Chromatographic separation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0585947A JPH0585947A (en) | 1993-04-06 |
| JP2938632B2 true JP2938632B2 (en) | 1999-08-23 |
Family
ID=17137232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3245681A Expired - Fee Related JP2938632B2 (en) | 1991-09-25 | 1991-09-25 | Chromatographic separation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2938632B2 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3746315B2 (en) | 1994-07-07 | 2006-02-15 | ダイセル化学工業株式会社 | Separating agent |
| JP4320065B2 (en) * | 1997-10-23 | 2009-08-26 | ダイセル化学工業株式会社 | Optical isomer separation method |
| US6428704B1 (en) * | 1998-08-07 | 2002-08-06 | Sekisui Chemical Co., Ltd. | Method for determination of hemoglobins |
| GB0606016D0 (en) * | 2006-03-25 | 2006-05-03 | Ionic Polymer Solutions Ltd | Quaternary ammonium compounds and their uses |
| CN106124678B (en) * | 2016-05-30 | 2017-09-05 | 中国水产科学研究院黄海水产研究所 | The quick screening method of perfluorochemical and its precursor substance in the flesh of fish |
| JP6802542B2 (en) * | 2016-09-02 | 2020-12-16 | 国立大学法人 和歌山大学 | Separation and purification method for cinnamon acid geometric isomers |
-
1991
- 1991-09-25 JP JP3245681A patent/JP2938632B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0585947A (en) | 1993-04-06 |
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