WO2016190043A1 - Method for analyzing saccharide - Google Patents

Method for analyzing saccharide Download PDF

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Publication number
WO2016190043A1
WO2016190043A1 PCT/JP2016/063200 JP2016063200W WO2016190043A1 WO 2016190043 A1 WO2016190043 A1 WO 2016190043A1 JP 2016063200 W JP2016063200 W JP 2016063200W WO 2016190043 A1 WO2016190043 A1 WO 2016190043A1
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eluent
base
inorganic salt
column
analyzing
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PCT/JP2016/063200
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French (fr)
Japanese (ja)
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修弥 中島
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昭和電工株式会社
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Priority to JP2017520581A priority Critical patent/JP6675391B2/en
Publication of WO2016190043A1 publication Critical patent/WO2016190043A1/en

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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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the present invention relates to a method for analyzing saccharides by liquid chromatography.
  • Liquid chromatography is widely used for the analysis of saccharides.
  • the saccharides are analyzed by ion exchange chromatography (IEC) using an ion exchange chromatography column, ligand exchange chromatography (LEC) using a ligand exchange chromatography column, and hydrophilic interaction chromatography.
  • IEC ion exchange chromatography
  • LEC ligand exchange chromatography
  • HILIC hydrophilic interaction chromatography
  • a strong alkaline aqueous solution is used as an eluent (see, for example, Non-Patent Document 1).
  • This method has high separation performance of saccharides, but there are problems such as decomposition of saccharides with a strong alkali aqueous solution and alkali resistance required for the analyzer. Further, the above-described method using a column for ligand exchange chromatography (see, for example, Patent Document 1) requires a high temperature condition, and therefore there is a problem that metal ions in the column filler are easily desorbed.
  • a column using hydrophilic interaction chromatography specifically, a column (hereinafter referred to as “amino column”) filled with a filler formed by chemically bonding an amino group to a substrate.
  • the method for analyzing saccharides using the hydrophilic interaction chromatography used is excellent not only in that it does not require an alkaline solution as an eluent but also in that it can be analyzed with good reproducibility at low temperatures (for example, Patent Document 2). reference).
  • saccharides are separated by utilizing the difference in hydrophilicity of saccharides.
  • sugars are very similar in structure to each other, despite being a diverse group of compounds.
  • glucose and galactose differ only in the bonding direction of one of five hydroxyl groups, and the other has a common structure. Because of the similar structure, there are saccharides that are very difficult to separate only by the difference in hydrophilicity.
  • the present invention has been made in view of the above circumstances. It is an object of the present invention to provide a method for analyzing saccharides by liquid chromatography, which can accurately separate saccharides that were difficult to separate by conventional methods.
  • the present inventors have completed the present invention. That is, by adding a base and an inorganic salt to the eluent and adjusting the pH and salt concentration of the eluent, it is possible to accurately separate saccharides that were difficult to separate by conventional methods by liquid chromatography. I found it.
  • the present invention provides a method for analyzing saccharides by liquid chromatography using a column for hydrophilic interaction chromatography, which comprises using an eluent containing a base and an inorganic salt.
  • the first aspect of the present invention is the following analysis method. [1] A step of preparing a column for hydrophilic interaction chromatography, an eluent containing a base and an inorganic salt, and a step of performing hydrophilic interaction chromatography using the column and the eluent. A method for analyzing saccharides.
  • the first aspect of the present invention preferably has the following features.
  • the base is sodium hydroxide or potassium hydroxide.
  • the inorganic salt is an alkali metal halide or an alkaline earth metal halide.
  • the combination of the base and the inorganic salt is any one of sodium hydroxide and potassium hydroxide, sodium chloride, potassium chloride and lithium chloride. Is one of the inorganic salts.
  • the filler used in the hydrophilic interaction chromatography column is polyvinyl alcohol in which an amino group is chemically bonded.
  • the concentrations of the base and the inorganic salt contained in the eluent are 1 mmol / L to 20 mmol / L, respectively.
  • the pH of the eluent containing the base and the inorganic salt is 11.0 or more and 13.0 or less.
  • the eluent contains water and an organic solvent, and a ratio of the organic solvent in the eluent is 50% or more and 95% or less.
  • FIG. 1 It is a schematic diagram which shows an example of the liquid chromatograph used with the analysis method of saccharides of this invention.
  • 2 is a chromatogram obtained in Example 1.
  • 2 is a chromatogram obtained in Comparative Example 2.
  • Hydrophilic interaction chromatography is generally a method for retaining and separating hydrophilic compounds using a stationary phase having a polar group and a mobile phase based on a combination of an organic solvent such as acetonitrile and water. Separation takes place by hydrophilic interactions.
  • the sample injected into the column elutes in order from a compound having low hydrophilicity (low polarity compound). More specifically, a hydrated phase is formed on the surface of the stationary phase (filler), and the sample is separated between the hydrated phase and the mobile phase. For this reason, polar compounds with high affinity are easily retained in the hydrated phase and elutes slowly.
  • the saccharide analysis method of the present invention is a saccharide analysis method in which analysis is performed by liquid chromatography using a hydrophilic interaction chromatography column, and an eluent containing a base and an inorganic salt is used.
  • the saccharides to be analyzed in the present invention can be selected as necessary, but mainly monosaccharides and oligosaccharides are analyzed.
  • the present invention is particularly effective for analysis of aldoses having an aldehyde group among these sugars. The amount of sample introduced is selected as necessary.
  • the column for hydrophilic interaction chromatography (hereinafter abbreviated as “column”) used in the present invention is arbitrarily selected, but is preferably a column packed with a filler described later.
  • the type, size and amount of the filler can be arbitrarily selected.
  • the column preferably used in the present invention is generally composed of, for example, a housing having a cylindrical shape and the like, and a filler filled in the housing, for example, a filler described later.
  • the material, size (inner diameter, length, etc.) and shape of the housing are not particularly limited and can be arbitrarily selected. Examples of preferred shapes include hollow cylindrical shapes and tube shapes.
  • You may have a cover part which has a flow hole for putting in or discharging
  • the flow hole functions as an entrance or an exit.
  • the lids may be integrated with the housing or may be detachable.
  • a commercially available column for hydrophilic interaction chromatography can be used.
  • the filler can be arbitrarily selected, and a filler having a base material such as silica gel or polymer and a functional group such as an ion exchange group or a polar group chemically bonded to the base material can be preferably used.
  • a filler having a base material such as silica gel or polymer and a functional group such as an ion exchange group or a polar group chemically bonded to the base material
  • polyvinyl alcohol is preferably used as the substrate that can be used in the present invention, and an amino group is preferably used as the functional group.
  • the filler is more preferably polyvinyl alcohol having an amino group chemically bonded thereto.
  • the amino group chemically bonded to the substrate is preferably an amino group having excellent hydrophilicity.
  • an amino group include a substrate such as polyvinyl alcohol, D-glucamine, N-methyl-D-glucamine, 1-amino-1-deoxy-D-mannitol, 1-amino-1 -Deoxy-D-galactitol, 1-amino-1-deoxy-D-iditol, 1-amino-1-deoxy-D-arabinitol, 1-amino-1-deoxy-D-xylitol, 4-amino-1, At least one selected from the group consisting of 2,3-butanetriol, 3-amino-1,2-propanediol, and 3-methylamino-1,2-propanediol is chemically bonded and introduced into the substrate And amino groups.
  • the particle size can be arbitrarily selected.
  • the volume average particle size is preferably 2.0 to 20.0 ⁇ m, more preferably 2.0 to 10.0 ⁇ m.
  • the specific surface area and the like can be arbitrarily selected.
  • 1.0 to 200 m 2 / g is preferable, and 50 to 150 m 2 / g is more preferable.
  • the average pore diameter can also be arbitrarily selected.
  • 1.0 to 20 nm is preferable, and 5 to 15 nm is more preferable.
  • the pore volume can also be arbitrarily selected.
  • 0.01 to 1.0 cm 3 / g is preferable, and 0.1 to 1.0 cm 3 / g is more preferable.
  • the eluent in the present invention contains a base and an inorganic salt. More specifically, the eluent in the present invention is preferably a mixed solution of an aqueous solution containing a base and an inorganic salt and an organic solvent. That is, in the eluent in the present invention, the contained solvent is a mixed solvent of water and an organic solvent.
  • the base in the present invention means an inorganic base exhibiting strong alkalinity.
  • an alkali metal hydroxide is preferable, and sodium hydroxide or potassium hydroxide is particularly preferable. These may be used alone or in combination.
  • the reason why sodium hydroxide or potassium hydroxide is preferable is that they are highly soluble in water and exhibit strong alkalinity. Similar to sodium hydroxide and potassium hydroxide, calcium hydroxide, magnesium hydroxide, and lithium hydroxide exhibiting strong alkalinity have low solubility in a solvent and may be precipitated in the eluent.
  • the inorganic salt in the present invention means a neutral inorganic salt.
  • Such an inorganic salt is arbitrarily selected, but an alkali metal halide or an alkaline earth metal halide is preferably used. These may be used in combination.
  • the reason why an alkali metal halide or an alkaline earth metal halide is preferable as the inorganic salt is that the solubility in water is high and a hydrated layer effective for liquid-liquid separation is easily formed in the stationary phase. is there.
  • the alkali metal halide can be arbitrarily selected. If a specific example is given, sodium chloride, potassium chloride, lithium chloride, potassium iodide, etc. can be used preferably. These may be used alone or in any combination of two or more.
  • the reason why sodium chloride, potassium chloride, lithium chloride, potassium iodide and the like are preferable as the alkali metal halide is that the use of these compounds is effective in forming a more effective hydrated layer.
  • the alkaline earth metal halide can be arbitrarily selected. If a specific example is given, calcium chloride, magnesium chloride, etc. can be used preferably. These may be used alone or in any combination of two or more. The reason why calcium chloride and magnesium chloride are preferable as the alkaline earth metal halide is that the use of these compounds is effective in forming a more effective hydrated layer.
  • the organic solvent is arbitrarily selected, but an organic solvent that is arbitrarily compatible with water is preferable.
  • Preferred examples of such an organic solvent include acetonitrile, tetrahydrofuran, acetone, ethanol, and methanol. These organic solvents may be used individually by 1 type, and 2 or more types may be mixed and used for them.
  • the ratio of the organic solvent in the eluent when mixing the aqueous solution containing the base and the inorganic salt with the organic solvent and a preferable range of the ratio are arbitrarily selected as necessary. It can.
  • the lower limit value can be selected as necessary, such as 50%, 55%, 60%, 70%, or 80%.
  • the upper limit value can also be arbitrarily selected, and examples thereof include 80%, 85%, 90%, and 95%.
  • the ratio of the organic solvent when mixing the organic solvent with the aqueous solution containing the base and the inorganic salt is The ratio is preferably 50% or more.
  • the ratio of the organic solvent is more preferably 70% or more and 95% or less by volume ratio.
  • the ratio of the organic solvent may be 50% or more and 95% or less, or 60% or more and 85% or less, as necessary.
  • an eluent having a high organic solvent ratio as described above is preferably used.
  • the base or the inorganic salt may be precipitated when an aqueous solution containing the base and the inorganic salt is mixed with the organic solvent.
  • the precipitated base or inorganic salt may clog the inside of the pipe or the column, leading to an increase in analytical pressure, deterioration of the column, or the like. Therefore, it is preferable to use a base or inorganic salt having sufficient solubility in a solvent.
  • Such a base or inorganic salt preferably has a water solubility of 30 g / 100 mL or more, more preferably 70 g / 100 mL or more, and even more preferably 100 g / 100 mL or more.
  • the solubility of sodium hydroxide in water is 109 g / 100 mL
  • the solubility of potassium hydroxide in water is 110 g / 100 mL
  • the solubility of sodium chloride in water is 35.7 g / 100 mL
  • the solubility of lithium chloride in water is 76.9 g /
  • the solubility of 100 mL and potassium iodide in water is 148 g / 100 mL.
  • the concentrations of the base and the inorganic salt contained in the eluent can be arbitrarily selected, but are preferably 1 mmol / L to 20 mmol / L, respectively.
  • the sum of the concentrations of the base and the inorganic salt contained in the eluent is preferably 20 mmol / L or less.
  • the mixing ratio of the base and the inorganic salt can be arbitrarily selected, but the molar ratio is preferably 5: 1 to 1: 1. More preferably, it is 3: 1 to 1: 1, and further preferably 2: 1 to 1: 1.
  • the mixing ratio of the base and the inorganic salt is within the above range, the retention time of each saccharide can be adjusted, and each saccharide can be separated satisfactorily.
  • the pH of the aqueous solution containing a base and an inorganic salt can be arbitrarily selected. It is preferably 10.0 or more, and more preferably 11.0 or more and 13.0 or less. By setting the pH of the aqueous solution containing the base and the inorganic salt to 10.0 or more, it is possible to prevent the base or the inorganic salt from being precipitated in the eluent prepared using the aqueous solution containing the base and the inorganic salt.
  • a liquid chromatograph 100 shown in FIG. 1 includes a container 110, a liquid pump 120, a sample introduction unit 130, along a direction (flow direction of the eluent 200) in which the eluent 200 as a mobile phase flows.
  • a column 140 and a detector 150 are sequentially provided. These containers, members, and apparatuses are connected to each other via a flow path 160 made of piping or the like.
  • An eluent 200 is accommodated in the container 110.
  • an eluent containing the above base and inorganic salt is used as the eluent 200.
  • the liquid flow pump 120 can send the eluent 200 in the container 110 at a constant flow rate.
  • the sample introduction unit 130 is provided in the middle of the flow path 160 that connects the liquid pump 120 and the column 140. A sample to be analyzed is introduced from the sample introduction unit 130 to the eluent 200 flowing through the flow channel 160.
  • Column 140 is a column in which a housing is filled with a filler formed by chemically bonding an amino group to the above-described base material.
  • the column 140 separates saccharides contained in the sample.
  • the detector 150 detects the saccharide separated in the column 140.
  • the sample introduction unit 130, the column 140, and the detector 150 may be installed in a thermostat not shown.
  • the thermostatic bath has a temperature control function and controls the temperature of the sample introduction unit 130, the column 140, and the detector 150. Thereby, stable analysis can be performed.
  • the control temperature can be set arbitrarily.
  • a sample is injected from the sample introduction unit 130 into the flow path 170, and the sample is added to the column 140 by the eluent 200 passed through the flow path 160 by the liquid flow pump 120. Led to.
  • the sample is preferably added in the form of a solution containing the substance to be detected.
  • the saccharide in the sample is separated, and the separated saccharide is measured by the detector 150 as a change amount from the signal when only the eluent 200 is used.
  • a data processing unit (not shown) connected to the detector 150 converts the output signal from the detector 150 into a chromatogram.
  • the eluent 200 that is, the flow rate of the mobile phase (the speed of flowing in the column 140) is not particularly limited, and can be arbitrarily selected as necessary. For example, a flow rate of 0.5 mL / min to 1.5 mL / min can be preferably used.
  • the temperature of the column 140 at the time of passing a mobile phase is not specifically limited, It can select arbitrarily.
  • the lower limit of the preferable temperature range can be selected as necessary, and examples thereof include 20 ° C., 30 ° C., 35 ° C., and 40 ° C.
  • An upper limit can also be arbitrarily selected, and examples thereof include 70 ° C., 60 ° C., 55 ° C., and 50 ° C.
  • a specific preferable temperature range is 30 to 60 ° C., more preferably 30 to 50 ° C.
  • the method for analyzing saccharides of the present invention in the method for analyzing saccharides by liquid chromatography using a column for hydrophilic interaction chromatography, separation can be achieved in the conventional method by using an eluent containing a base and an inorganic salt.
  • the difficult saccharides can be separated with high accuracy so that the analysis peaks do not overlap with the peaks.
  • analysis with excellent quantification is possible.
  • a strong alkaline eluent is used.
  • the packing material which consists of a silica gel base material it is preferable to select a material, a member, and conditions in consideration of the possibility of column deterioration due to the eluent.
  • a base material resistant to strong alkalinity for example, by using a base material made of polyvinyl alcohol, it is possible to perform analysis with a long life and excellent stability.
  • Example 1 As a column, Shodex (registered trademark) HILICpak VG-50 4E manufactured by Showa Denko Co., Ltd. (column material: SUS, functional group: amino group, packing particle size: 5 ⁇ m, column inner diameter: 4.6 mm, column length: 150 mm) And a differential refractive index detector, five kinds of sugars of arabinose, xylose, mannose, galactose and glucose were analyzed. Specifically, an aqueous solution containing 0.1% of each of these five sugars was prepared and used as a sample. Shodex HILICpak VG-50 4E is a column packed with a packing material in which amino groups are chemically bonded to a base material made of polyvinyl alcohol.
  • acetonitrile and an aqueous solution containing 8 mM sodium hydroxide and 5 mM sodium chloride (1 mM 1 mmol / L) in a volume ratio of (acetonitrile): (8 mM sodium hydroxide and 5 mM sodium chloride)
  • a mixed solution containing 82:18 was prepared and used.
  • the flow rate of the eluent (the speed of the eluent flowing through the column) was 1.0 mL / min, and the column temperature was 40 ° C.
  • FIG. 2 The obtained chromatogram is shown in FIG.
  • 1 is an arabinose peak
  • 2 is a xylose peak
  • 3 is a mannose peak
  • 4 is a galactose peak
  • 5 is a glucose peak.
  • Table 1 shows the separation degree of saccharides in the chromatogram shown in FIG.
  • 1-2 is the resolution of peaks 1 and 2 in FIG. 2
  • 3-4 is the resolution of peaks 3 and 4 in FIG. 2
  • 4-5 is 4 and 5 in FIG.
  • the degree of peak separation is shown.
  • the resolution of the chromatogram peak is a numerical value indicating the degree of separation from the adjacent peak. For example, when peak 1 and peak 2 exist, it is calculated based on the following formula (1) or formula (2).
  • the horizontal axis indicates the retention time
  • the peak width at the distribution base line indicates the base width
  • the peak width at half the peak height is indicated as the half-value width.
  • Resolution 2 ⁇ [(Peak 2 retention time) ⁇ (Peak 1 retention time)] / [(Peak 1 base width) + (Peak 2 base width)] (1)
  • Resolution 1.18 ⁇ [(Retention time of peak 2) ⁇ (Retention time of peak 1)] / [(Half width of peak 1) + (Half width of peak 2)] (2)
  • the degree of separation was calculated based on the above equation (2).
  • the present invention contributes to a method for further improving the separation performance of saccharides in a saccharide analysis method by liquid chromatography that has already been widely used as an analysis technique for separating and quantifying a plurality of saccharides. Improvement of the resolution can contribute to shortening of analysis time by increasing the flow rate of the eluent and / or reducing consumption of the eluent by reducing the column diameter or shortening the column.
  • the present invention can provide a method for analyzing saccharides by liquid chromatography, which can accurately separate saccharides that were difficult to separate by conventional methods.

Abstract

A method for analyzing a saccharide by liquid chromatography, characterized by having a step for preparing a hydrophilic interaction chromatography column and an eluent including a base and an inorganic salt, and a step for performing hydrophilic interaction chromatography using the column and the eluent.

Description

糖類の分析方法Analysis method of saccharides
　本発明は、液体クロマトグラフィーによる糖類の分析方法に関する。
　本願は、２０１５年５月２８日に、日本に出願された特願２０１５－１０８１４５号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a method for analyzing saccharides by liquid chromatography.
This application claims priority based on Japanese Patent Application No. 2015-108145 filed in Japan on May 28, 2015, the contents of which are incorporated herein by reference.
　糖類の分析には、液体クロマトグラフィーが広く用いられている。糖類の分析方法としては、イオン交換クロマトグラフィー用カラムを用いるイオン交換クロマトグラフィー（ＩＥＣ）、配位子交換クロマトグラフィー用カラムを用いる配位子交換クロマトグラフィー（ＬＥＣ）、及び、親水性相互作用クロマトグラフィー用カラムを用いる親水性相互作用クロマトグラフィー（ＨＩＬＩＣ）等が挙げられる。
　イオン交換クロマトグラフィー用カラムを用いる前記方法では、溶離液として、強アルカリ水溶液が用いられる（例えば、非特許文献１参照）。この方法は、糖類の分離性能が高い反面、強アルカリ水溶液による糖類の分解や、分析装置にアルカリ耐性が求められる等の課題がある。
　また、配位子交換クロマトグラフィー用カラムを用いる前記方法（例えば、特許文献１参照）では、高温条件を必要とし、その為、カラムの充填剤の金属イオンが脱離しやすいという課題がある。 Liquid chromatography is widely used for the analysis of saccharides. The saccharides are analyzed by ion exchange chromatography (IEC) using an ion exchange chromatography column, ligand exchange chromatography (LEC) using a ligand exchange chromatography column, and hydrophilic interaction chromatography. And hydrophilic interaction chromatography (HILIC) using a chromatography column.
In the method using an ion exchange chromatography column, a strong alkaline aqueous solution is used as an eluent (see, for example, Non-Patent Document 1). This method has high separation performance of saccharides, but there are problems such as decomposition of saccharides with a strong alkali aqueous solution and alkali resistance required for the analyzer.
Further, the above-described method using a column for ligand exchange chromatography (see, for example, Patent Document 1) requires a high temperature condition, and therefore there is a problem that metal ions in the column filler are easily desorbed.
　これらの方法に対して、親水性相互作用クロマトグラフィーを用いた、具体的には基材にアミノ基を化学結合させてなる充填剤を充填したカラム（以下、「アミノカラム」と言う。）を用いた親水性相互作用クロマトグラフィーを用いた、糖類の分析方法は、溶離液としてアルカリ溶液を必要としないばかりではなく、低温において再現性よく分析できるという点で優れている（例えば、特許文献２参照）。 For these methods, a column using hydrophilic interaction chromatography, specifically, a column (hereinafter referred to as “amino column”) filled with a filler formed by chemically bonding an amino group to a substrate. The method for analyzing saccharides using the hydrophilic interaction chromatography used is excellent not only in that it does not require an alkaline solution as an eluent but also in that it can be analyzed with good reproducibility at low temperatures (for example, Patent Document 2). reference).
特開昭６１－７１３５４号公報JP-A-61-71354 特開平２－２２７１４４号公報JP-A-2-227144
　アミノカラムを用いた糖類の分析方法では、糖類の親水性の違いを利用して、糖類を分離する。しかしながら、糖類は、多様な化合物群であるにもかかわらず、互いの構造が非常に似ている。
例えば、グルコースとガラクトースは、５つある水酸基のうちの１つの結合の向きが異なるのみであり、その他は共通した構造を持つ。このように構造が似ている事から、親水性の違いだけで分離することが非常に難しい糖類がある。 In the saccharide analysis method using an amino column, saccharides are separated by utilizing the difference in hydrophilicity of saccharides. However, sugars are very similar in structure to each other, despite being a diverse group of compounds.
For example, glucose and galactose differ only in the bonding direction of one of five hydroxyl groups, and the other has a common structure. Because of the similar structure, there are saccharides that are very difficult to separate only by the difference in hydrophilicity.
　本発明は、上記事情に鑑みてなされたものである。従来法では分離が難しかった糖類を精度よく分離することができる、液体クロマトグラフィーによる糖類の分析方法を提供することを目的とする。 The present invention has been made in view of the above circumstances. It is an object of the present invention to provide a method for analyzing saccharides by liquid chromatography, which can accurately separate saccharides that were difficult to separate by conventional methods.
　本発明者等は、上記課題を解決するために鋭意研究を重ねた結果、本発明を完成するに至った。すなわち、溶離液に塩基と無機塩を添加して、溶離液のｐＨと塩濃度を調整することにより、液体クロマトグラフィーによって、従来法では分離が難しかった糖類を、精度よく分離することができることを見出した。 As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, by adding a base and an inorganic salt to the eluent and adjusting the pH and salt concentration of the eluent, it is possible to accurately separate saccharides that were difficult to separate by conventional methods by liquid chromatography. I found it.
　本発明は親水性相互作用クロマトグラフィー用カラムを用いた液体クロマトグラフィーによる糖類の分析方法であって、塩基と無機塩を含む溶離液を用いることを特徴とする糖類の分析方法を提供する。
　具体的には、本発明の第一の態様は以下の分析方法である。
［１］親水性相互作用クロマトグラフィー用カラムと、塩基と無機塩を含む溶離液とを用意する工程と、前記カラム及び前記溶離液を用いて、親水性相互作用クロマトグラフィーを行う工程とを有することを特徴とする糖類の分析方法。 The present invention provides a method for analyzing saccharides by liquid chromatography using a column for hydrophilic interaction chromatography, which comprises using an eluent containing a base and an inorganic salt.
Specifically, the first aspect of the present invention is the following analysis method.
[1] A step of preparing a column for hydrophilic interaction chromatography, an eluent containing a base and an inorganic salt, and a step of performing hydrophilic interaction chromatography using the column and the eluent. A method for analyzing saccharides.
本発明の第一の態様は、以下の特徴を有することが好ましい。
［２］　上記［１］の分析方法において、前記塩基は、水酸化ナトリウムまたは水酸化カリウムである。 The first aspect of the present invention preferably has the following features.
[2] In the analysis method of the above [1], the base is sodium hydroxide or potassium hydroxide.
［３］　上記［１］または［２］において、前記無機塩は、アルカリ金属のハロゲン化物またはアルカリ土類金属のハロゲン化物である。 [3] In the above [1] or [2], the inorganic salt is an alkali metal halide or an alkaline earth metal halide.
［４］　上記［１］～［３］のいずれかにおいて、前記塩基と前記無機塩との配合比が、モル比で、塩基：無機塩＝５：１～１：１である。 [4] In any one of the above [1] to [3], the mixing ratio of the base and the inorganic salt is a molar ratio of base: inorganic salt = 5: 1 to 1: 1.
［５］　上記［１］～［４］のいずれかにおいて、前記塩基と前記無機塩との組み合わせが、水酸化ナトリウムおよび水酸化カリウムのいずれか１つの塩基と、塩化ナトリウム、塩化カリウムおよび塩化リチウムのうちのいずれか１つの無機塩である。 [5] In any one of the above [1] to [4], the combination of the base and the inorganic salt is any one of sodium hydroxide and potassium hydroxide, sodium chloride, potassium chloride and lithium chloride. Is one of the inorganic salts.
［６］　上記［１］～［５］のいずれかにおいて、前記親水性相互作用クロマトグラフィー用カラムに用いる充填剤が、アミノ基が化学結合されたポリビニルアルコールである。 [6] In any one of the above [1] to [5], the filler used in the hydrophilic interaction chromatography column is polyvinyl alcohol in which an amino group is chemically bonded.
［７］　上記［１］～［６］のいずれかにおいて、前記溶離液に含まれる前記塩基と前記無機塩の濃度が、それぞれ１ｍｍｏｌ／Ｌ～２０ｍｍｏｌ／Ｌである。 [7] In any one of the above [1] to [6], the concentrations of the base and the inorganic salt contained in the eluent are 1 mmol / L to 20 mmol / L, respectively.
［８］　上記［１］～［７］のいずれかにおいて、前記塩基と前記無機塩を含む前記溶離液のｐＨが、１１．０以上１３．０以下である。
［９］　上記［１］～［８］のいずれかにおいて、前記溶離液が、水と有機溶媒を含み、溶離液中の有機溶媒の比率が５０％以上９５％以下である。 [8] In any one of the above [1] to [7], the pH of the eluent containing the base and the inorganic salt is 11.0 or more and 13.0 or less.
[9] In any one of the above [1] to [8], the eluent contains water and an organic solvent, and a ratio of the organic solvent in the eluent is 50% or more and 95% or less.
　本発明によれば、従来法では分離が難しかった糖類を精度よく分離することができる、液体クロマトグラフィーによる糖類の分析方法を提供することができる。 According to the present invention, it is possible to provide a method for analyzing saccharides by liquid chromatography, which can accurately separate saccharides that were difficult to separate by conventional methods.
本発明の糖類の分析方法で用いられる液体クロマトグラフの一例を示す模式図である。It is a schematic diagram which shows an example of the liquid chromatograph used with the analysis method of saccharides of this invention. 実施例１で得られたクロマトグラムである。2 is a chromatogram obtained in Example 1. 比較例２で得られたクロマトグラムである。2 is a chromatogram obtained in Comparative Example 2.
　本発明の糖類の分析方法の好ましい実施の形態の例について説明する。
　なお、本実施の形態の例は、発明の趣旨をより良く理解させるために具体的に説明するものであり、特に指定のない限り、本発明を限定するものではない。本発明の範囲内において、必要に応じて、変更、省略、交換及び／又は追加することも可能である。 The example of preferable embodiment of the analysis method of the saccharide | sugar of this invention is demonstrated.
In addition, the example of the present embodiment is specifically described for better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified. Changes, omissions, replacements and / or additions may be made as necessary within the scope of the present invention.
[親水性相互作用クロマトグラフィー]
　親水性相互作用クロマトグラフィーは、一般に、極性基を有する固定相と、アセトニトリル等の有機溶媒及び水の組み合わせの系による移動相とを用いて、親水性化合物を保持及び分離する方法である。親水性相互作用によって分離が行われる。カラムに注入されたサンプルは、親水性の低い化合物（低極性化合物）から順に溶出する。より具体的には、固定相（充填剤）の表面に水和相が形成され、水和相と移動相の間で試料の分離が行われる。この為、水和相には親和性の高い極性化合物が保持されやすく遅く溶出する。一方、極性が低い化合物は保持されにくく、早い段階で溶出する。本発明は、親水性相互作用クロマトグラフィーにおいて、優れた効果を供することが可能である。
［糖類の分析方法］
　本発明の糖類の分析方法は、親水性相互作用クロマトグラフィー用カラムを用いた液体クロマトグラフィーにより分析を行う、糖類の分析方法であって、塩基と無機塩を含む溶離液を用いる。
　本発明において分析される糖類は、必要に応じて選択できるが、主に単糖とオリゴ糖が分析される。本発明は、これらの糖のうち、アルデヒド基を持つアルドースの分析に特に有効である。試料の導入量は必要に応じて選択される。 [Hydrophilic interaction chromatography]
Hydrophilic interaction chromatography is generally a method for retaining and separating hydrophilic compounds using a stationary phase having a polar group and a mobile phase based on a combination of an organic solvent such as acetonitrile and water. Separation takes place by hydrophilic interactions. The sample injected into the column elutes in order from a compound having low hydrophilicity (low polarity compound). More specifically, a hydrated phase is formed on the surface of the stationary phase (filler), and the sample is separated between the hydrated phase and the mobile phase. For this reason, polar compounds with high affinity are easily retained in the hydrated phase and elutes slowly. On the other hand, compounds with low polarity are not easily retained and elute at an early stage. The present invention can provide an excellent effect in hydrophilic interaction chromatography.
[Method for analyzing sugars]
The saccharide analysis method of the present invention is a saccharide analysis method in which analysis is performed by liquid chromatography using a hydrophilic interaction chromatography column, and an eluent containing a base and an inorganic salt is used.
The saccharides to be analyzed in the present invention can be selected as necessary, but mainly monosaccharides and oligosaccharides are analyzed. The present invention is particularly effective for analysis of aldoses having an aldehyde group among these sugars. The amount of sample introduced is selected as necessary.
「カラム」
　本発明で用いられる親水性相互作用クロマトグラフィー用カラム（以下、「カラム」と略す。）は任意に選択されるが、後述する充填剤が充填されたカラムであることが好ましい。充填剤の種類やサイズや量も任意に選択できる。本発明で好ましく使用されるカラムは、例えば、円筒形状等の形状を有するハウジングと、そのハウジングの内部に充填された充填剤、例えば、後述する充填剤、とから、概略構成されている。
　ハウジングの材質、大きさ（内径、長さ等）及び形状は、特に限定されず、任意に選択できる。好ましい形状の例を挙げれば、中空の円筒形状やチューブ形状等を挙げる事ができる。ハウジングの両端部又は片端部には、液を入れる又は排出する為の流通孔を有する蓋部を有しても良い。前記流通孔は入り口又は出口として機能する。蓋部はそれぞれハウジングと一体になっていても、または着脱可能であっても良い。本発明は、一般に販売されている親水性相互作用クロマトグラフィー用カラムを使用することができる。 "column"
The column for hydrophilic interaction chromatography (hereinafter abbreviated as “column”) used in the present invention is arbitrarily selected, but is preferably a column packed with a filler described later. The type, size and amount of the filler can be arbitrarily selected. The column preferably used in the present invention is generally composed of, for example, a housing having a cylindrical shape and the like, and a filler filled in the housing, for example, a filler described later.
The material, size (inner diameter, length, etc.) and shape of the housing are not particularly limited and can be arbitrarily selected. Examples of preferred shapes include hollow cylindrical shapes and tube shapes. You may have a cover part which has a flow hole for putting in or discharging | emitting a liquid in the both ends or one end part of a housing. The flow hole functions as an entrance or an exit. The lids may be integrated with the housing or may be detachable. In the present invention, a commercially available column for hydrophilic interaction chromatography can be used.
「充填剤」
　充填剤は任意で選択でき、シリカゲルやポリマー等の基材と、この基材に化学結合されたイオン交換基や極性基のような官能基と、を有する充填剤を好ましく使用できる。本発明で使用できる基材としては、例えば、ポリビニルアルコールが好ましく用いられ、官能基としては、アミノ基が好ましく用いられる。本発明において、充填剤としては、アミノ基が化学結合されたポリビニルアルコールがより好ましい。 "filler"
The filler can be arbitrarily selected, and a filler having a base material such as silica gel or polymer and a functional group such as an ion exchange group or a polar group chemically bonded to the base material can be preferably used. For example, polyvinyl alcohol is preferably used as the substrate that can be used in the present invention, and an amino group is preferably used as the functional group. In the present invention, the filler is more preferably polyvinyl alcohol having an amino group chemically bonded thereto.
　基材に化学結合されたアミノ基は、親水性に優れたアミノ基であることが好ましい。このようなアミノ基の具体例を挙げれば、基材に、例えばポリビニルアルコールに、Ｄ－グルカミン、Ｎ－メチル－Ｄ－グルカミン、１－アミノ－１－デオキシ－Ｄ－マンニトール、１－アミノ－１－デオキシ－Ｄ－ガラクチトール、１－アミノ－１－デオキシ－Ｄ－イジトール、１－アミノ－１－デオキシ－Ｄ－アラビニトール、１－アミノ－１－デオキシ－Ｄ－キシリトール、４－アミノ－１，２，３－ブタントリオール、３－アミノ－１，２－プロパンジオール、及び３－メチルアミノ－１，２－プロパンジオールからなる群から選択される少なくとも１つを化学結合させて、基材に導入された、アミノ基が挙げられる。
　充填剤が粒子形状を有する場合、その粒径は任意に選択することができる。例を挙げれば体積平均粒径が２．０～２０．０μｍであることが好ましく、２．０～１０．０μｍがより好ましい。比表面積なども任意に選択することができ、例を挙げれば、１．０～２００ｍ２/ｇが好ましく、５０～１５０ｍ２/ｇがより好ましい。平均細孔径も任意に選択でき、例を挙げれば１．０～２０ｎｍが好ましく、５～１５ｎｍがより好ましい。細孔容積も任意に選択でき、例を挙げれば０．０１～１．０ｃｍ３/ｇが好ましく、０．１～１．０ｃｍ３/ｇがより好ましい。 The amino group chemically bonded to the substrate is preferably an amino group having excellent hydrophilicity. Specific examples of such an amino group include a substrate such as polyvinyl alcohol, D-glucamine, N-methyl-D-glucamine, 1-amino-1-deoxy-D-mannitol, 1-amino-1 -Deoxy-D-galactitol, 1-amino-1-deoxy-D-iditol, 1-amino-1-deoxy-D-arabinitol, 1-amino-1-deoxy-D-xylitol, 4-amino-1, At least one selected from the group consisting of 2,3-butanetriol, 3-amino-1,2-propanediol, and 3-methylamino-1,2-propanediol is chemically bonded and introduced into the substrate And amino groups.
When the filler has a particle shape, the particle size can be arbitrarily selected. For example, the volume average particle size is preferably 2.0 to 20.0 μm, more preferably 2.0 to 10.0 μm. The specific surface area and the like can be arbitrarily selected. For example, 1.0 to 200 m 2 / g is preferable, and 50 to 150 m 2 / g is more preferable. The average pore diameter can also be arbitrarily selected. For example, 1.0 to 20 nm is preferable, and 5 to 15 nm is more preferable. The pore volume can also be arbitrarily selected. For example, 0.01 to 1.0 cm 3 / g is preferable, and 0.1 to 1.0 cm 3 / g is more preferable.
「溶離液」
　本発明における溶離液は、塩基および無機塩を含む。より詳細には、本発明における溶離液は、塩基および無機塩を含む水溶液と、有機溶媒との混合溶液であることが好ましい。すなわち、本発明における溶離液では、含まれる溶媒が、水と有機溶媒の混合溶媒からなる。 "Eluent"
The eluent in the present invention contains a base and an inorganic salt. More specifically, the eluent in the present invention is preferably a mixed solution of an aqueous solution containing a base and an inorganic salt and an organic solvent. That is, in the eluent in the present invention, the contained solvent is a mixed solvent of water and an organic solvent.
　本発明における塩基とは、強アルカリ性を示す無機塩基を意味する。このような無機塩基は任意に選択されるが、具体例を挙げれば、アルカリ金属の水酸化物が好ましく、特に水酸化ナトリウムまたは水酸化カリウムが好ましい。これらは単独で使用しても、組み合わせて使用しても良い。水酸化ナトリウムまたは水酸化カリウムが好ましい理由は、これらは水に対する溶解度が高く、かつ、強アルカリ性を示すからである。
　なお、水酸化ナトリウムおよび水酸化カリウムと同様に、強アルカリ性を示す水酸化カルシウム、水酸化マグネシウム、及び水酸化リチウムは、溶媒に対する溶解度が低く、溶離液中に析出することがある。このため、これら化合物の本発明における効果は、前記２つの化合物（水酸化ナトリウムおよび水酸化カリウム）ほどは大きくない。また、アミン化合物は、塩基性度が低いため、本発明の効果が、前記２つの化合物ほどは大きくない。 The base in the present invention means an inorganic base exhibiting strong alkalinity. Although such an inorganic base is arbitrarily selected, an alkali metal hydroxide is preferable, and sodium hydroxide or potassium hydroxide is particularly preferable. These may be used alone or in combination. The reason why sodium hydroxide or potassium hydroxide is preferable is that they are highly soluble in water and exhibit strong alkalinity.
Similar to sodium hydroxide and potassium hydroxide, calcium hydroxide, magnesium hydroxide, and lithium hydroxide exhibiting strong alkalinity have low solubility in a solvent and may be precipitated in the eluent. For this reason, the effects of these compounds in the present invention are not as great as those of the two compounds (sodium hydroxide and potassium hydroxide). Further, since the amine compound has a low basicity, the effect of the present invention is not as great as that of the two compounds.
　本発明における無機塩とは、中性の無機塩を意味する。このような無機塩は任意に選択されるが、アルカリ金属のハロゲン化物またはアルカリ土類金属のハロゲン化物が好ましく使用される。これらは組み合わせて使用しても良い。無機塩として、アルカリ金属のハロゲン化物またはアルカリ土類金属のハロゲン化物が好ましい理由は、水に対する溶解度が高く、かつ、固定相において、液液分離に有効な水和層が形成しやすくなるからである。
　アルカリ金属のハロゲン化物は任意に選択できる。具体例を挙げれば、塩化ナトリウム、塩化カリウム、塩化リチウム、及びヨウ化カリウム等が好ましく使用できる。これらは単独で使用しても、２種類以上を任意で組み合わせて使用しても良い。アルカリ金属のハロゲン化物として、塩化ナトリウム、塩化カリウム、塩化リチウム、及びヨウ化カリウム等が好ましい理由は、これら化合物を使用することがより効果的な水和層を形成することに有効であるからである。
　アルカリ土類金属のハロゲン化物は任意に選択できる。具体例を挙げれば、塩化カルシウム、及び塩化マグネシウム等が好ましく使用できる。これらは単独で使用しても、２種類以上を任意で組み合わせて使用しても良い。アルカリ土類金属のハロゲン化物として、塩化カルシウム、及び塩化マグネシウム等が好ましい理由は、これら化合物を使用することがより効果的な水和層を形成することに有効であるからである。 The inorganic salt in the present invention means a neutral inorganic salt. Such an inorganic salt is arbitrarily selected, but an alkali metal halide or an alkaline earth metal halide is preferably used. These may be used in combination. The reason why an alkali metal halide or an alkaline earth metal halide is preferable as the inorganic salt is that the solubility in water is high and a hydrated layer effective for liquid-liquid separation is easily formed in the stationary phase. is there.
The alkali metal halide can be arbitrarily selected. If a specific example is given, sodium chloride, potassium chloride, lithium chloride, potassium iodide, etc. can be used preferably. These may be used alone or in any combination of two or more. The reason why sodium chloride, potassium chloride, lithium chloride, potassium iodide and the like are preferable as the alkali metal halide is that the use of these compounds is effective in forming a more effective hydrated layer. is there.
The alkaline earth metal halide can be arbitrarily selected. If a specific example is given, calcium chloride, magnesium chloride, etc. can be used preferably. These may be used alone or in any combination of two or more. The reason why calcium chloride and magnesium chloride are preferable as the alkaline earth metal halide is that the use of these compounds is effective in forming a more effective hydrated layer.
　有機溶媒は任意に選択されるが、水と任意に相溶するものが好ましい。このような有機溶媒としては、例えば、アセトニトリル、テトラヒドロフラン、アセトン、エタノール、及びメタノール等が好ましく挙げられる。これらの有機溶媒は、１種を単独で用いてもよく、２種以上を混合して用いてもよい。 The organic solvent is arbitrarily selected, but an organic solvent that is arbitrarily compatible with water is preferable. Preferred examples of such an organic solvent include acetonitrile, tetrahydrofuran, acetone, ethanol, and methanol. These organic solvents may be used individually by 1 type, and 2 or more types may be mixed and used for them.
　本発明における溶離液において、塩基および無機塩を含む水溶液と、有機溶媒とを混合する際の、有機溶媒の溶離液中に占める比率や、前記比率の好ましい範囲は、必要に応じて任意に選択できる。例えば、下限値は、５０％、５５％、６０％、７０％又は８０％など必要に応じて選択できる。上限値も任意に選択でき、例えば、８０％、８５％、９０％又は９５％などを挙げる事ができる。
糖類の保持時間が長すぎることがなく、また、分離性能を高めることが要求される場合には、塩基および無機塩を含む水溶液と、有機溶媒とを混合する際の有機溶媒の比率は、体積比で５０％以上であることが好ましい。また、糖類の保持時間が長すぎることがなく、また、分離性能をさらに高めることが要求される場合には、有機溶媒の比率は、体積比で７０％以上９５％以下であることがより好ましい。有機溶媒の比率は、必要に応じて、５０％以上９５％以下であったり、６０％以上８５％以下などであっても良い。 In the eluent of the present invention, the ratio of the organic solvent in the eluent when mixing the aqueous solution containing the base and the inorganic salt with the organic solvent, and a preferable range of the ratio are arbitrarily selected as necessary. it can. For example, the lower limit value can be selected as necessary, such as 50%, 55%, 60%, 70%, or 80%. The upper limit value can also be arbitrarily selected, and examples thereof include 80%, 85%, 90%, and 95%.
When the retention time of the saccharide is not too long and it is required to improve the separation performance, the ratio of the organic solvent when mixing the organic solvent with the aqueous solution containing the base and the inorganic salt is The ratio is preferably 50% or more. In addition, when the retention time of the saccharide is not too long and it is required to further improve the separation performance, the ratio of the organic solvent is more preferably 70% or more and 95% or less by volume ratio. . The ratio of the organic solvent may be 50% or more and 95% or less, or 60% or more and 85% or less, as necessary.
　本発明では、上記のように有機溶媒の比率が高い溶離液が好ましく用いられる。この時、溶媒に対する溶解度が低い塩基または無機塩を用いると、塩基および無機塩を含む水溶液と、有機溶媒とを混合した際、塩基または無機塩が析出する可能性がある。析出した塩基または無機塩を含む溶離液を用いると、その析出した塩基または無機塩により、配管内やカラム内が詰り、分析圧の上昇や、カラムの劣化等の原因となる可能性がある。そのため、塩基や無機塩は、溶媒に対して充分な溶解性を有するものを用いることが好ましい。このような塩基や無機塩は、水に対する溶解度が３０ｇ／１００ｍＬ以上であることが好ましく、７０ｇ／１００ｍＬ以上であることがより好ましく、１００ｇ／１００ｍＬ以上であることがさらに好ましい。
　なお、水酸化ナトリウムの水に対する溶解度は１０９ｇ／１００ｍＬ、水酸化カリウムの水に対する溶解度は１１０ｇ／１００ｍＬ、塩化ナトリウムの水に対する溶解度は３５．７ｇ／１００ｍＬ、塩化リチウムの水に対する溶解度は７６．９ｇ／１００ｍＬ、及びヨウ化カリウムの水に対する溶解度は１４８ｇ／１００ｍＬである。 In the present invention, an eluent having a high organic solvent ratio as described above is preferably used. At this time, if a base or an inorganic salt having low solubility in a solvent is used, the base or the inorganic salt may be precipitated when an aqueous solution containing the base and the inorganic salt is mixed with the organic solvent. When an eluent containing a precipitated base or inorganic salt is used, the precipitated base or inorganic salt may clog the inside of the pipe or the column, leading to an increase in analytical pressure, deterioration of the column, or the like. Therefore, it is preferable to use a base or inorganic salt having sufficient solubility in a solvent. Such a base or inorganic salt preferably has a water solubility of 30 g / 100 mL or more, more preferably 70 g / 100 mL or more, and even more preferably 100 g / 100 mL or more.
The solubility of sodium hydroxide in water is 109 g / 100 mL, the solubility of potassium hydroxide in water is 110 g / 100 mL, the solubility of sodium chloride in water is 35.7 g / 100 mL, and the solubility of lithium chloride in water is 76.9 g / The solubility of 100 mL and potassium iodide in water is 148 g / 100 mL.
　また、本発明における溶離液において、塩基または無機塩の濃度が高過ぎると、塩基または無機塩が溶離液中に析出するおそれがある。そのため、溶離液に含まれる塩基と無機塩の濃度は任意に選択できるが、それぞれ１ｍｍｏｌ／Ｌ～２０ｍｍｏｌ／Ｌであることが好ましい。溶離液に含まれる塩基と無機塩の濃度の和は、２０ｍｍｏｌ／Ｌ以下であることが好ましい。 In the eluent in the present invention, if the concentration of the base or inorganic salt is too high, the base or inorganic salt may be precipitated in the eluent. Therefore, the concentrations of the base and the inorganic salt contained in the eluent can be arbitrarily selected, but are preferably 1 mmol / L to 20 mmol / L, respectively. The sum of the concentrations of the base and the inorganic salt contained in the eluent is preferably 20 mmol / L or less.
　また、本発明における溶離液において、塩基と無機塩の配合比は任意に選択できるが、モル比で、５：１～１：１が好ましい。より好ましくは、３：１～１：１であり、更に好ましくは２：１～１：１である。溶離液において、塩基と無機塩の配合比を前記の範囲内とすることにより、各糖類の保持時間を調節でき、各糖類が良好に分離可能である。 In the eluent of the present invention, the mixing ratio of the base and the inorganic salt can be arbitrarily selected, but the molar ratio is preferably 5: 1 to 1: 1. More preferably, it is 3: 1 to 1: 1, and further preferably 2: 1 to 1: 1. In the eluent, when the mixing ratio of the base and the inorganic salt is within the above range, the retention time of each saccharide can be adjusted, and each saccharide can be separated satisfactorily.
　また、塩基および無機塩を含む水溶液のｐＨは任意に選択することができる。１０．０以上であることが好ましく、１１．０以上１３．０以下であることがより好ましい。
　塩基および無機塩を含む水溶液のｐＨを１０．０以上とすることにより、この塩基および無機塩を含む水溶液を用いて調製した溶離液において、塩基または無機塩が析出することを防止できる。 Moreover, the pH of the aqueous solution containing a base and an inorganic salt can be arbitrarily selected. It is preferably 10.0 or more, and more preferably 11.0 or more and 13.0 or less.
By setting the pH of the aqueous solution containing the base and the inorganic salt to 10.0 or more, it is possible to prevent the base or the inorganic salt from being precipitated in the eluent prepared using the aqueous solution containing the base and the inorganic salt.
　本発明の糖類の分析方法では、例えば、図１に示すような液体クロマトグラフが用いられる。
　図１に示す液体クロマトグラフ１００は、移動相としての溶離液２００が流される方向（溶離液２００の通液方向）に沿って、容器１１０と、通液ポンプ１２０と、試料導入部１３０と、カラム１４０と、検出器１５０と、を順に備えている。これら容器や部材や装置は、配管等からなる流路１６０を介して互いに連結されている。 In the saccharide analysis method of the present invention, for example, a liquid chromatograph as shown in FIG. 1 is used.
A liquid chromatograph 100 shown in FIG. 1 includes a container 110, a liquid pump 120, a sample introduction unit 130, along a direction (flow direction of the eluent 200) in which the eluent 200 as a mobile phase flows. A column 140 and a detector 150 are sequentially provided. These containers, members, and apparatuses are connected to each other via a flow path 160 made of piping or the like.
　容器１１０内には、溶離液２００が収容されている。
　溶離液２００としては、上記の塩基および無機塩を含む溶離液が用いられる。 An eluent 200 is accommodated in the container 110.
As the eluent 200, an eluent containing the above base and inorganic salt is used.
　通液ポンプ１２０は、容器１１０内の溶離液２００を一定の流量で送ることができる。
　試料導入部１３０は、通液ポンプ１２０とカラム１４０を連結する流路１６０の途中に設けられている。この試料導入部１３０から、流路１６０を流れる溶離液２００に、分析の対象となる試料が導入される。 The liquid flow pump 120 can send the eluent 200 in the container 110 at a constant flow rate.
The sample introduction unit 130 is provided in the middle of the flow path 160 that connects the liquid pump 120 and the column 140. A sample to be analyzed is introduced from the sample introduction unit 130 to the eluent 200 flowing through the flow channel 160.
　カラム１４０は、ハウジングに、上記の基材にアミノ基を化学結合させてなる充填剤が充填されたカラムである。カラム１４０は、試料に含まれる、糖類を分離する。 Column 140 is a column in which a housing is filled with a filler formed by chemically bonding an amino group to the above-described base material. The column 140 separates saccharides contained in the sample.
　検出器１５０は、カラム１４０にて分離された糖類を検出する。
　また、液体クロマトグラフ１００では、試料導入部１３０、カラム１４０および検出器１５０が、図示しない恒温漕内に設置されていてもよい。恒温漕は、温度制御機能を備え、試料導入部１３０、カラム１４０および検出器１５０の温度を制御する。これにより、安定した分析を行うことができる。なお制御温度は任意に設定することができる。 The detector 150 detects the saccharide separated in the column 140.
In the liquid chromatograph 100, the sample introduction unit 130, the column 140, and the detector 150 may be installed in a thermostat not shown. The thermostatic bath has a temperature control function and controls the temperature of the sample introduction unit 130, the column 140, and the detector 150. Thereby, stable analysis can be performed. The control temperature can be set arbitrarily.
　液体クロマトグラフ１００を用いた分析では、試料導入部１３０から流路１７０内に試料が注入され、前記試料は、通液ポンプ１２０によって流路１６０内を通液される溶離液２００により、カラム１４０へと導かれる。試料は、検出すべき物質を含んだ溶液の状態で加えられることが好ましい。カラム１４０にて、試料中の糖類が分離され、分離された糖類を、検出器１５０が、溶離液２００のみの場合の信号からの変化量として計測する。検出器１５０からの出力信号を、検出器１５０に接続された図示しないデータ処理部がクロマトグラムに変換する。 In the analysis using the liquid chromatograph 100, a sample is injected from the sample introduction unit 130 into the flow path 170, and the sample is added to the column 140 by the eluent 200 passed through the flow path 160 by the liquid flow pump 120. Led to. The sample is preferably added in the form of a solution containing the substance to be detected. In the column 140, the saccharide in the sample is separated, and the separated saccharide is measured by the detector 150 as a change amount from the signal when only the eluent 200 is used. A data processing unit (not shown) connected to the detector 150 converts the output signal from the detector 150 into a chromatogram.
　溶離液２００、すなわち、移動相の流速（カラム１４０内を流れる速さ）は、特に限定されず、必要に応じて任意に選択できる。例えば、０．５ｍＬ／ｍｉｎ～１．５ｍＬ／ｍｉｎの流速を好ましく使用することができる。
　また、移動相を通液する際のカラム１４０の温度は、特に限定されず、任意に選択できる。好ましい温度範囲の下限は、必要に応じて選択でき、例えば、２０℃、３０℃、３５℃、又は４０℃などを挙げる事ができる。上限も任意に選択でき、７０℃、６０℃、５５℃、又は５０℃などを挙げる事ができる。具体的な好ましい温度範囲を挙げれば、３０℃～６０℃であることが好ましく、３０～５０℃であることがより好ましい。 The eluent 200, that is, the flow rate of the mobile phase (the speed of flowing in the column 140) is not particularly limited, and can be arbitrarily selected as necessary. For example, a flow rate of 0.5 mL / min to 1.5 mL / min can be preferably used.
Moreover, the temperature of the column 140 at the time of passing a mobile phase is not specifically limited, It can select arbitrarily. The lower limit of the preferable temperature range can be selected as necessary, and examples thereof include 20 ° C., 30 ° C., 35 ° C., and 40 ° C. An upper limit can also be arbitrarily selected, and examples thereof include 70 ° C., 60 ° C., 55 ° C., and 50 ° C. A specific preferable temperature range is 30 to 60 ° C., more preferably 30 to 50 ° C.
　本発明の糖類の分析方法によれば、親水性相互作用クロマトグラフィー用カラムを用いた液体クロマトグラフィーによる糖類の分析方法において、塩基と無機塩を含む溶離液を用いることにより、従来法では分離が難しかった糖類を、精度よく、分析ピークとピークが重ならないように、分離することができる。その結果、定量性に優れた分析が可能である。なお、本発明の糖類の分析方法によれば、強アルカリ性の溶離液が用いられる。このため、シリカゲル基材からなる充填剤を用いる場合、溶離液によるカラムの劣化の可能性を考慮して、材料や部材や条件を選択する事が好ましい。例えば、基材として強アルカリ性に対して耐性のある基材を使用することにより、例えば、ポリビニルアルコールからなる基材を用いることにより、長寿命で安定性に優れた分析が可能である。 According to the method for analyzing saccharides of the present invention, in the method for analyzing saccharides by liquid chromatography using a column for hydrophilic interaction chromatography, separation can be achieved in the conventional method by using an eluent containing a base and an inorganic salt. The difficult saccharides can be separated with high accuracy so that the analysis peaks do not overlap with the peaks. As a result, analysis with excellent quantification is possible. According to the method for analyzing saccharides of the present invention, a strong alkaline eluent is used. For this reason, when using the packing material which consists of a silica gel base material, it is preferable to select a material, a member, and conditions in consideration of the possibility of column deterioration due to the eluent. For example, by using a base material resistant to strong alkalinity as the base material, for example, by using a base material made of polyvinyl alcohol, it is possible to perform analysis with a long life and excellent stability.
　以下、実施例および比較例により本発明をさらに具体的に説明するが、本発明は以下の実施例のみに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but the present invention is not limited to the following examples.
［実施例１］
　カラムとして、昭和電工社製のＳｈｏｄｅｘ（登録商標）　ＨＩＬＩＣｐａｋ　ＶＧ－５０　４Ｅ（カラム材質：ＳＵＳ、官能基：アミノ基、充填剤粒径：５μｍ、カラム内径：４．６ｍｍ、カラム長さ：１５０ｍｍ）と、示差屈折率検出器とを用いて、アラビノース、キシロース、マンノース、ガラクトース、グルコースの５種の糖を分析した。具体的には、これら５つの糖をそれぞれ０．１％含む水溶液を用意し、試料として用いた。　Ｓｈｏｄｅｘ　ＨＩＬＩＣｐａｋ　ＶＧ－５０　４Ｅは、ポリビニルアルコールからなる基材にアミノ基を化学結合させた充填剤を充填したカラムである。
　溶離液としては、アセトニトリルと、８ｍＭの水酸化ナトリウムおよび５ｍＭの塩化ナトリウムを含む水溶液（１ｍＭ＝１ｍｍｏｌ／Ｌ）とを、体積比で、（アセトニトリル）：（８ｍＭの水酸化ナトリウムおよび５ｍＭの塩化ナトリウムを含む水溶液）＝８２：１８の割合で含む、混合溶液を用意し用いた。なお、８ｍＭの水酸化ナトリウムおよび５ｍＭの塩化ナトリウムを含む溶液においては、水酸化ナトリウム：塩化ナトリウム＝８：５（モル比）である。
　溶離液の流速（カラム内を流れる溶離液の速さ）を１．０ｍＬ／ｍｉｎ、カラム温度を４０℃とした。
　得られたクロマトグラムを図２に示す。図２において、１はアラビノースのピーク、２はキシロースのピーク、３はマンノースのピーク、４はガラクトースのピーク、及び５はグルコースのピークを示す。また、図２に示すクロマトグラムにおける糖類の分離度を表１に示す。表１において、１－２は、図２における１と２のピークの分離度、３－４は、図２における３と４のピークの分離度、及び４－５は、図２における４と５のピークの分離度を示す。
　なお、クロマトグラムのピークの分離度は、隣接するピークからの分離の程度を表す数値である。例えば、ピーク１とピーク２が存在する場合、下記の式（１）または式（２）に基づいて算出される。図２や図３においては、横軸が保持時間を示し、分布のベースラインにおけるピーク幅がベース幅を示し、ピーク高さの半分でのピーク幅を半値幅として示す。
　分離度＝２×［（ピーク２の保持時間）－（ピーク１の保持時間）］／［（ピーク１のベース幅）+（ピーク２のベース幅）］・・・（１）
　分離度＝１．１８×［（ピーク２の保持時間）－（ピーク１の保持時間）］／［（ピーク１の半値幅）+（ピーク２の半値幅）］・・・（２）
　本実施例では、上記の式（２）に基づいて、分離度を算出した。 [Example 1]
As a column, Shodex (registered trademark) HILICpak VG-50 4E manufactured by Showa Denko Co., Ltd. (column material: SUS, functional group: amino group, packing particle size: 5 μm, column inner diameter: 4.6 mm, column length: 150 mm) And a differential refractive index detector, five kinds of sugars of arabinose, xylose, mannose, galactose and glucose were analyzed. Specifically, an aqueous solution containing 0.1% of each of these five sugars was prepared and used as a sample. Shodex HILICpak VG-50 4E is a column packed with a packing material in which amino groups are chemically bonded to a base material made of polyvinyl alcohol.
As an eluent, acetonitrile and an aqueous solution containing 8 mM sodium hydroxide and 5 mM sodium chloride (1 mM = 1 mmol / L) in a volume ratio of (acetonitrile): (8 mM sodium hydroxide and 5 mM sodium chloride) A mixed solution containing 82:18 was prepared and used. In a solution containing 8 mM sodium hydroxide and 5 mM sodium chloride, the ratio is sodium hydroxide: sodium chloride = 8: 5 (molar ratio).
The flow rate of the eluent (the speed of the eluent flowing through the column) was 1.0 mL / min, and the column temperature was 40 ° C.
The obtained chromatogram is shown in FIG. In FIG. 2, 1 is an arabinose peak, 2 is a xylose peak, 3 is a mannose peak, 4 is a galactose peak, and 5 is a glucose peak. In addition, Table 1 shows the separation degree of saccharides in the chromatogram shown in FIG. In Table 1, 1-2 is the resolution of peaks 1 and 2 in FIG. 2, 3-4 is the resolution of peaks 3 and 4 in FIG. 2, and 4-5 is 4 and 5 in FIG. The degree of peak separation is shown.
The resolution of the chromatogram peak is a numerical value indicating the degree of separation from the adjacent peak. For example, when peak 1 and peak 2 exist, it is calculated based on the following formula (1) or formula (2). In FIGS. 2 and 3, the horizontal axis indicates the retention time, the peak width at the distribution base line indicates the base width, and the peak width at half the peak height is indicated as the half-value width.
Resolution = 2 × [(Peak 2 retention time) − (Peak 1 retention time)] / [(Peak 1 base width) + (Peak 2 base width)] (1)
Resolution = 1.18 × [(Retention time of peak 2) − (Retention time of peak 1)] / [(Half width of peak 1) + (Half width of peak 2)] (2)
In this example, the degree of separation was calculated based on the above equation (2).
［比較例１］
　溶離液として、アセトニトリルと、８ｍＭの水酸化ナトリウム水溶液とを、体積比で、アセトニトリル：８ｍＭの水酸化ナトリウム水溶液＝８２：１８の割合で含む混合溶液を用いた以外は、実施例１と同様にして、上記の５種の糖を分析した。
　得られたクロマトグラムに基づく糖類の分離度を表１に示す。 [Comparative Example 1]
The same procedure as in Example 1 was used, except that a mixed solution containing acetonitrile and an 8 mM aqueous sodium hydroxide solution in a volume ratio of acetonitrile: 8 mM aqueous sodium hydroxide = 82: 18 was used as an eluent. The above five sugars were analyzed.
Table 1 shows the degree of separation of saccharides based on the obtained chromatogram.
Figure JPOXMLDOC01-appb-T000001
 　　　　　　　　　　　　　　　　　　
Figure JPOXMLDOC01-appb-T000001
 　　　　　　　　　　　　　　　　　　
　一般に、クロマトグラムのピークの分離度が１．５以上の場合、隣接するピーク同士が完全に分離していると判断されている。一方、クロマトグラムのピークの分離度が１．５未満の場合、隣接するピークが一部重なっていると判断される。
　表１の結果から、実施例１では、全てのピークが完全に分離していることが分かった。
一方、比較例１では、３－４の分離度のみ１．５よりも低いことが分かった。以上の結果から、塩基（水酸化ナトリウム）を含み、無機塩（塩化ナトリウム）を含まない混合溶液を溶離液として用いた場合、上記の５種の糖を全て完全に分離できないことが分かった。 Generally, when the degree of separation of chromatogram peaks is 1.5 or more, it is determined that adjacent peaks are completely separated. On the other hand, when the resolution of the chromatogram peak is less than 1.5, it is determined that adjacent peaks partially overlap.
From the results in Table 1, it was found that in Example 1, all peaks were completely separated.
On the other hand, in Comparative Example 1, it was found that only the separation degree of 3-4 was lower than 1.5. From the above results, it was found that when the mixed solution containing a base (sodium hydroxide) and not containing an inorganic salt (sodium chloride) was used as an eluent, all of the above five sugars could not be completely separated.
［比較例２］
　溶離液として、アセトニトリルと、５ｍＭの塩化ナトリウム水溶液とを、体積比で、アセトニトリル：５ｍＭの塩化ナトリウム水溶液＝８２：１８の割合で含む混合溶液を用いた以外は、実施例１と同様にして、上記の５種の糖を分析した。
　得られたクロマトグラムを図３に示す。図３において、１はアラビノースのピーク、２はキシロースのピーク、３はマンノースのピーク、４はガラクトースのピーク、及び５はグルコースのピークを示す。
　図３の結果から、１と２のピークが重なっているとともに、４と５のピークが重なっていることが分かった。すなわち、無機塩（塩化ナトリウム）を含み、塩基（水酸化ナトリウム）を含まない混合溶液を溶離液として用いた場合、上記の５種の糖を全て完全に分離できないことが分かった。そのため、比較例２では、分離度を測定することができなかった。 [Comparative Example 2]
Except that a mixed solution containing acetonitrile and a 5 mM sodium chloride aqueous solution in a volume ratio of acetonitrile: 5 mM sodium chloride aqueous solution = 82: 18 was used as an eluent in the same manner as in Example 1, The above five sugars were analyzed.
The obtained chromatogram is shown in FIG. In FIG. 3, 1 is an arabinose peak, 2 is a xylose peak, 3 is a mannose peak, 4 is a galactose peak, and 5 is a glucose peak.
From the results of FIG. 3, it was found that the peaks of 1 and 2 overlap and the peaks of 4 and 5 overlap. That is, when a mixed solution containing an inorganic salt (sodium chloride) and no base (sodium hydroxide) was used as an eluent, it was found that not all of the above five sugars could be completely separated. Therefore, in Comparative Example 2, the degree of separation could not be measured.
　本発明は、複数の糖類を分離し定量する分析手法として、すでに広く用いられている液体クロマトグラフィーによる糖類の分析方法において、糖類の分離性能をさらに改善する方法に寄与する。分離能の改善は、溶離液の高流速化による分析時間の短縮、及び／又は、カラムの小径化や短尺化による溶離液の消費量の削減に寄与できる。
　本発明は、従来法では分離が難しかった糖類を精度よく分離することができる、液体クロマトグラフィーによる糖類の分析方法を提供できる。 INDUSTRIAL APPLICABILITY The present invention contributes to a method for further improving the separation performance of saccharides in a saccharide analysis method by liquid chromatography that has already been widely used as an analysis technique for separating and quantifying a plurality of saccharides. Improvement of the resolution can contribute to shortening of analysis time by increasing the flow rate of the eluent and / or reducing consumption of the eluent by reducing the column diameter or shortening the column.
The present invention can provide a method for analyzing saccharides by liquid chromatography, which can accurately separate saccharides that were difficult to separate by conventional methods.
１００・・・液体クロマトグラフ
１１０・・・容器
１２０・・・通液ポンプ
１３０・・・試料導入部
１４０・・・カラム
１５０・・・検出器
１６０・・・流路
２００・・・溶離液 DESCRIPTION OF SYMBOLS 100 ... Liquid chromatograph 110 ... Container 120 ... Fluid pump 130 ... Sample introduction part 140 ... Column 150 ... Detector 160 ... Channel 200 ... Eluent

Claims (9)

  1. 　親水性相互作用クロマトグラフィー用カラムと、塩基と無機塩を含む溶離液と、を用意する工程と、
    　前記カラム及び前記溶離液を用いて、親水性相互作用クロマトグラフィーを行う工程と、
    を有することを特徴とする、液体クロマトグラフィーによる糖類の分析方法。     Preparing a hydrophilic interaction chromatography column and an eluent containing a base and an inorganic salt;
    Performing hydrophilic interaction chromatography using the column and the eluent;
    A method for analyzing saccharides by liquid chromatography, comprising:
  2. 　前記塩基は、水酸化ナトリウムまたは水酸化カリウムであることを特徴とする、請求項１に記載の糖類の分析方法。 The method for analyzing a saccharide according to claim 1, wherein the base is sodium hydroxide or potassium hydroxide.
  3. 　前記無機塩は、アルカリ金属のハロゲン化物またはアルカリ土類金属のハロゲン化物であることを特徴とする、請求項１または２に記載の糖類の分析方法。 3. The method for analyzing saccharides according to claim 1, wherein the inorganic salt is an alkali metal halide or an alkaline earth metal halide.
  4. 　前記塩基と前記無機塩との配合比が、モル比で、塩基：無機塩＝５：１～１：１である、請求項１～３のいずれか１項に記載の糖類の分析方法。 The method for analyzing saccharides according to any one of claims 1 to 3, wherein a mixing ratio of the base and the inorganic salt is a molar ratio of base: inorganic salt = 5: 1 to 1: 1.
  5. 　前記塩基と前記無機塩との組み合わせが、水酸化ナトリウムおよび水酸化カリウムのいずれか１つの塩基と、塩化ナトリウム、塩化カリウムおよび塩化リチウムのうちのいずれか１つの無機塩との組み合わせである、請求項１～４のいずれか１項に記載の糖類の分析方法。 The combination of the base and the inorganic salt is a combination of any one of sodium hydroxide and potassium hydroxide and an inorganic salt of any one of sodium chloride, potassium chloride, and lithium chloride. Item 5. The method for analyzing saccharides according to any one of Items 1 to 4.
  6. 　前記親水性相互作用クロマトグラフィー用カラムに用いる充填剤が、アミノ基が化学結合されたポリビニルアルコールである、請求項１～５のいずれか１項に記載の糖類の分析方法。 The method for analyzing saccharides according to any one of claims 1 to 5, wherein the filler used in the column for hydrophilic interaction chromatography is polyvinyl alcohol having an amino group chemically bonded thereto.
  7. 　前記溶離液に含まれる前記塩基と前記無機塩の濃度が、それぞれ１ｍｍｏｌ／Ｌ～２０ｍｍｏｌ／Ｌである、請求項１～６のいずれか１項に記載の糖類の分析方法。 The method for analyzing saccharides according to any one of claims 1 to 6, wherein the concentrations of the base and the inorganic salt contained in the eluent are 1 mmol / L to 20 mmol / L, respectively.
  8. 　前記塩基と前記無機塩を含む前記溶離液のｐＨが、１１．０以上１３．０以下である、請求項１～７のいずれか１項に記載の糖類の分析方法。 The method for analyzing saccharides according to any one of claims 1 to 7, wherein the pH of the eluent containing the base and the inorganic salt is 11.0 or more and 13.0 or less.
  9. 　前記溶離液が、水と有機溶媒を含み、溶離液中の有機溶媒の比率が５０％以上９５％以下である、請求項１～８のいずれか１項に記載の糖類の分析方法。 The method for analyzing saccharides according to any one of claims 1 to 8, wherein the eluent contains water and an organic solvent, and the ratio of the organic solvent in the eluent is 50% or more and 95% or less.
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